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First published online September 7, 2007
doi: 10.1242/10.1242/dev.010298

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Pattern formation during de novo assembly of the Arabidopsis shoot meristem

¹ Division of Biology, California Institute of Technology, Pasadena, CA, USA.
² Department of Botany and Plant Sciences, University of California, Riverside, CA, USA.
³ Laboratorie RDP, Ecole Normale Superieur de Lyon, Lyon, France.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Overview of the de novo shoot induction system. (A) Root explants were harvested from 2-week-old seedlings and (B) transferred to auxin-rich CIM, which induces cell proliferation, resulting in (C) callus formation. (D) Transfer of callus to cytokinin-rich SIM induces greening and induction of shoot meristems from callus often in clusters (marked by two green leaves).

View larger version (128K): [in this window] [in a new window]   Fig. 2. Hormone response and gene expression during callus induction. All samples were stained with propidium iodide (red) to stain cell walls. (A) In wild-type roots, the auxin-responsive reporter, pDR5rev::3XVENUS-N7 (green), was present in a subset of cells in the root vasculature, lateral root progenitors and columellar root cap cells. (B) Clusters of small cells marked by pDR5rev::3XVENUS-N7 reporter (green) proliferate to form callus, 5 days after induction on CIM. (C) After 8 days of CIM induction, the pDR5rev::3XVENUS-N7 reporter was weakly expressed in callus. (D) Pre-CIM cytokinin-responsive pARR5::GFP reporter expression (green) in the root stele, and lateral root progenitors. (E) pARR5::GFP reporter expression, 8 days and (F) 2 weeks after CIM induction, was visible in proliferating callus cells. (G) Pre-CIM pCUC2::3XVENUS-N7 reporter expression (green) in a subset of cells within the root stele and lateral root meristems. (H) pCUC2::3XVENUS-N7 reporter expression 8 days and (I) 2 weeks after CIM induction marked proliferating callus cells originating from sites of lateral root formation, root meristems and pericycle. (J,K) Two weeks induction on CIM without cytokinin, resulted in cell proliferation and expression of the pCUC2::3XVENUS-N7 (J) and pARR5::GFP reporters (K). (L) Two weeks after induction on CIM without 2,4-D, callus was not induced and expression of the pCUC2::3XVENUS-N7 reporter was faint and confined to the vasculature of the primary root. Scale bars: 50 µm (A,B,D,E,G,H); 100 µm (C,F,I-L). Arrowheads indicate lateral roots.

View larger version (88K): [in this window] [in a new window]   Fig. 3. Partition of gene expression and cell identity within callus. (A) Mounds of small, dividing cells marked by the pCUC2::3XVENUS-N7 reporter (green) formed and (B) gave rise to new shoot meristems (arrowheads), often observed in clusters. Chlorophyll autofluorescence is in red. (C,D) Scanning electron micrographs of early regenerating meristems (arrowheads, C), and (D) a late stage regenerated shoot emerging from callus. (E) The pWUS::mGFP-ER reporter (green) was expressed in callus cells poorly stained by FM4-64 dye (red) following 5 days induction on SIM. (F,G) Shoot progenitors (F, arrowhead, 12 days on SIM) were labeled with FM4-64 dye, and emerged from regions with peripheral pWUS::mGFP-ER expression, and formed mature shoot meristems (G), also strongly stained by FM4-64 dye. The pWUS::mGFP-ER reporter was upregulated in the center of the developing meristem (arrowhead). (H) pCUC2::3XVENUS-N7 (green) and pWUS::DsRed-N7 (red) reporters were active in opposing domains of cells, sometimes in gradients, shown after 10 days on SIM. (I) Higher magnification after 11 days on SIM, showing clusters of cells expressing the CUC2 reporter (arrowheads) surrounded by pWUS::DsRed-N7 expressing cells. (J) At later stages, WUS::DsRed-N7 expression was initiated in the center of the mound of shoot progenitors while pCUC2::3XVENUS-N7 was restricted to the future peripheral zone, shown here after 12 days on SIM. Scale bars: 50 µm (A,C,I,J); 100 µm (B,E-H); 300 µm (D). Pn, primordia.

View larger version (97K): [in this window] [in a new window]   Fig. 4. Partition of hormone response within callus. (A) The pARR5::GFP reporter (green) was active in callus forming shoot meristems (arrowhead), but downregulated in primordia [incipient primordia (I)1 and primordia (P)1]. Chlorophyll autofluorescence is in red. (B) Regenerating meristems (arrowheads) marked by pPIN1::PIN1-GFP (green) expression, emerged from callus with low pDR5rev::3XVENUS-N7 (red) expression, while pDR5rev::3XVENUS-N7 signal was observed in peripheral callus and within initiating primordia flanking meristems. (C) pPIN1::PIN1-GFP reporter expression (green) within the shoot progenitors. (D,E) Strong pDR5rev::3XVENUS-N7 expression (blue) was later detected during the initiation (D) and outgrowth of organ primordia (P1-P3; E). Chlorophyll autofluorescence is in red. (F) pDR5::3XVENUS-N7 signal (red) was not observed 1 minute after application of auxin paste (arrows) to callus initiating shoot meristems (arrowhead), marked by pPIN1::PIN1-GFP (green). (G) Four hours after application of auxin paste (arrows) pDR5::3XVENUS- N7 signal (red) was observed throughout the callus in contrast to the meristem itself (arrowhead), in which pDR5::3XVENUS-N7 signal was only in developing primordia. Scale bars: 50 µm (A,C-E); 100 µm (B,F,G).

View larger version (121K): [in this window] [in a new window]   Fig. 5. Pattern formation within the shoot promeristem. (A) pPIN1::PIN1-GFP expression (green) was upregulated in the superficial layer of shoot meristem progenitor cells marked by pCUC2::3XVENUS-N7 reporter expression (red), and in labeled primordium initials (I1 and I2). (B) After 24 hours, primordium initials grew into primordia (P1 and P2) and pCUC2::3XVENUS-N7 (red) was expressed in the meristem boundaries. (C) PIN1-GFP protein was (green) polarized towards the apex of the shoot progenitors (arrows) and away from peripheral cells marked by the pWUS::DsRed-N7 reporter (blue). (D) Early pREV::REV-VENUS expression (red) was observed in the center of the progenitors underneath the pPIN1::PIN1-GFP (green) domain. Chlorophyll autofluorescence is in blue. (E) 24 hours later in the same developing meristem, pREV::REV-VENUS expression (red) was expressed in the adaxial sides of initiating primordia (I1 and I2), and (F) was similarly expressed in primordia within later stage shoot meristems. (G-I) pFIL::DsRed-N7 expression (red) was upregulated in areas flanking the early pPIN1::PIN1-GFP (green) domain (G) and was later upregulated on the abaxial side of early primordia (H) and older primordia (I). (J) pSTM::STM-VENUS (blue) was expressed in a ring surrounding shoot progenitors and a subset of cells within the promeristem (11 days on SIM) while local pPIN1::PIN1-GFP reporter (green) upregulation marked sites of primordium initiation (I1 and I2). pWUS::DsRed-N7 reporter (red) was expressed in peripheral cells and upregulated in the center of the developing meristem. (K,L) 24 hours later in the same shoot progenitors, pSTM::STM-VENUS (blue) was upregulated within the meristem between the developing primordia (P1 and P2; K) and was maintained through 48 hours of imaging during which primordia grew and two new primordia were initiated (I1 and I2; L). (M) pCLV3::mGFP5-ER expression (green) was absent from shoot progenitors marked by pPIN1::PIN1-CFP expression (white) and peripheral cells marked by the pWUS::DsRed-N7 reporter (red). (N) pCLV3::mGFP5-ER expression (green) was detected after primordial outgrowth from the periphery of the developing meristem. (O) pCLV3::mGFP5-ER expression (green) was also observed in later stage shoot meristems which expressed a p35S::YFP 29-1 transgene (yellow). Scale bars: 50 µm (A,B,D-O); 5 µm (C).

View larger version (80K): [in this window] [in a new window]   Fig. 6. L1 layer specification and development of meristem structure. (A) pML1::GFP5-ER reporter (green) was upregulated in a subset of superficial shoot meristem progenitors, marked by the pCUC2::3XVENUS-N7 (red) marker. (B,C) Expression of the pML1::GFP5-ER reporter (green) was L1 specific within the shoot progenitors (B) but not L1 specific in cross-sections of callus (C). (D) 24 hours later, pML1::GFP5-ER expression was upregulated in the shoot progenitors. (E) 72 hours later, pML1::GFP5-ER expression was homogeneously expressed within the L1 of the meristem as primordia (I1 and P1) were initiated. (F) 96 hours later, two early primordia (P1 and P2) were evident. (G) pPIN1::PIN1-GFP (green) and pSTM::STM-VENUS (red) were upregulated with similar timing in small patches of cells marked by the ubiquitous pRIBO::2XCFP-N7 marker (blue). (H) 24 hours later, pPIN1::PIN1-GFP marked initiating primordia (I1, I2). (I) After 48 hours, primordia (P1 and P2) labeled by pPIN1::PIN1-GFP have grown outwards and pSTM::STM-VENUS was expressed in the developing meristem. (J) After 72 hours of observation, meristems derived from small numbers of initial cells gave rise to fewer primordia than meristems with more cells. Scale bars: 50 µm (A-J).

View larger version (53K): [in this window] [in a new window]   Fig. 7. WUS and PIN1 are functionally required for efficient shoot meristem induction. (A) Bar graph showing average numbers of shoots formed after 4 weeks of induction on SIM from 2 cm callus explants in wild type (Ler) versus wus-1, and in a separate experiment, Ler versus pin1-4.(B) Bar graph showing number of shoot promeristems, marked by pPIN1::PIN1-GFP and pSTM::STM-VENUS coexpression, in Ler versus wus-1. (C) pPIN1::PIN1-GFP (green) and pSTM::STM-VENUS (blue) expression in a shoot promeristem initiated in the wus-1 mutant. Chlorophyll autofluorescence is in red. (D) 24 hours later, the shoot promeristem initiated early primordia (P1 and P2) marked by pPIN1::PIN1-GFP, while pSTM::STM-VENUS marked the presumptive shoot meristem. (E) 48 hours later, primordia (P1 and P2) have further developed and two early primordia initials (I1 and I2) have formed near the apex of the meristem. Scale bars: 50 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 8. Schematic of de novo shoot meristem organization from callus. Auxin-rich CIM (A) induces proliferation of multipotent cells in the root leading to callus formation (B). (C) Transfer to cytokinin-rich SIM induces partition of cell identity and behavior within callus marked by the CUC2 (yellow) and WUS (red) reporters. Arrowhead indicates shoot progenitors. (D) Clusters of CUC2-labeled shoot progenitors proliferate among neighboring WUS expressing (red lines) non-progenitor cells in areas of high cytokinin and low auxin response. (E) 24-48 hours later, PIN1 and ML1 reporters (both green) are upregulated within the superficial layer of the shoot promeristem while STM (blue) is upregulated in a ring of surrounding cells and within the promeristem. Within the membrane of shoot progenitors, PIN1 protein is directed towards the apex of the promeristem (arrows), and thus is predicted to transport auxin into the promeristem from surrounding cells. (F) 48-96 hours later, PIN1 becomes locally upregulated within the peripheral zone and marks sites of primordial initiation. PIN1 protein becomes locally polarized towards sites of primordia formation (arrows). FIL (magenta) is expressed in the abaxial sides of newly initiated primordia. CLV3 expression (teal) is initiated within the central zone after WUS expression (red) initiates within the center of the meristem. pSTM::STM-VENUS is expressed within the meristem.

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