PLETHORA transcription factors orchestrate de novo organ patterning during Arabidopsis lateral root outgrowth

Abstract

Plant development is characterized by repeated initiation of meristems, regions of dividing cells that give rise to new organs. During lateral root (LR) formation, new LR meristems are specified to support the outgrowth of LRs along a new axis. The determination of the sequential events required to form this new growth axis has been hampered by redundant activities of key transcription factors. Here, we characterize the effects of three PLETHORA (PLT) transcription factors, PLT3, PLT5, and PLT7, during LR outgrowth. In plt3plt5plt7 triple mutants, the morphology of lateral root primordia (LRP), the auxin response gradient, and the expression of meristem/tissue identity markers are impaired from the "symmetry-breaking" periclinal cell divisions during the transition between stage I and stage II, wherein cells first acquire different identities in the proximodistal and radial axes. Particularly, PLT1, PLT2, and PLT4 genes that are typically expressed later than PLT3, PLT5, and PLT7 during LR outgrowth are not induced in the mutant primordia, rendering "PLT-null" LRP. Reintroduction of any PLT clade member in the mutant primordia completely restores layer identities at stage II and rescues mutant defects in meristem and tissue establishment. Therefore, all PLT genes can activate the formative cell divisions that lead to de novo meristem establishment and tissue patterning associated with a new growth axis.

Keywords: axis formation; branching; cell specification; meristem; plant architecture.

Fig. 1.

Critical requirement for three PLT genes in formative periclinal cell divisions. (A–D) Confocal images and corresponding cell outlines of WT LRP at indicated stages stained with propidium iodide: (A) stage I, (B) stage II, (C) stage III, and (D) emerged. (E) Schematic representation of auxin response (on the left) and tissue specificity (on the right) in the primary root meristem of WT. (F–I) Confocal images and corresponding cell outlines of plt3plt5plt7 LRP at indicated stages stained with propidium iodide: (F) stage I, (G) stage II, (H) stage III, and (I) emerged. Circles indicate LRP central cells without PeD. Arrows indicate abnormal cell division planes. (J–L) Data quantification details are described in Materials and Methods. (J) Periclinal cell division (PeD) counts in LRP central cell files at different stages in pPLT3::GUS marked WT and plt3plt5plt7 roots at 7 d postgermination (d.p.g.) from left to right: WT LRP at stage II (S II), WT LRP older than stage II (≥III), plt3plt5plt7 LRP at stage II (S II), and plt3plt5plt7 LRP older than stage II (≥III). (K) Stage proportion of LRP/LR in pPLT3::GUS marked WT and plt3plt5plt7 roots at 7 d.p.g. (n = 20). *P < 0.05 (Student’s t test); **P < 0.01 (Student’s t test). (L) Number of newly initiated LRP in pPLT3::GUS marked WT and plt3plt5plt7 roots at 7 d.p.g. (n = 20). **P < 0.01 (Student’s t test). CEI, cortex/endodermis initial; COL, columella; Cor, cortex; Endo, endodermis; Epi, epidermis; LRC, lateral root cap. (Scale bars: 100 μm.)

Fig. 2.

Auxin response gradually delocalizes, and PLT1, PLT2, and PLT4 promoters are not activated in plt3plt5plt7 LRP. (A and B) Confocal images of DR5::GFP in LRP at indicated stages: (A) WT and (B) plt3plt5plt7. (C and D) Confocal images of PIN1:GFP in LRP at indicated stages: (C) WT and (D) plt3plt5plt7. (E and F) Confocal images of PIN3:GFP in LRP at indicated stages: (E) WT and (F) plt3plt5plt7. (G and H) Confocal images of pPLT1::erCFP in LRP at indicated stages: (G) WT and (H) plt3plt5plt7. (I and J) Confocal images of pPLT2::erCFP in LRP at indicated stages: (I) WT and (J) plt3plt5plt7. (K and L) Confocal images of pPLT4::erCFP in LRP at indicated stages: (K) WT and (L) plt3plt5plt7. Asterisks indicate stage I LRP. Arrows indicate earliest morphological stages of detectable marker expression during LR outgrowth. Triangles indicate cell layers in LRP at stage II/III. Arrowheads indicate LRP central cells without periclinal cell division. (Scale bar: 100 μm.)

Fig. 3.

The expression of key cell fate regulators is disrupted in plt3plt5plt7 LRP. (A and B) Confocal images of SHR:GFP in LRP at indicated stages: (A) WT and (B) plt3plt5plt7. (C and D) Confocal images of pSCR::H2B:YFP in LRP at indicated stages: (C) WT and (D) plt3plt5plt7. (E and F) Confocal images of pWOX5::GFP in LRP at indicated stages: (E) WT and (F) plt3plt5plt7. (G and H) Confocal images of pFEZ::GFP:MBD in LRP at indicated stages: (G) WT and (H) plt3plt5plt7. (I and J) Confocal images of SMB:GFP in LRP at indicated stages: (I) WT and (J) plt3plt5plt7. (K and L) Confocal images of pWER::erCFP in LRP at indicated stages: (K) WT and (L) plt3plt5plt7. Asterisks indicate stage I LRP. Arrows indicate earliest morphological stages of detectable marker expression during LR outgrowth. Triangles indicate cell layers in LRP at stage II/III. Arrowheads indicate LRP central cells without periclinal cell division. (Scale bar: 100 μm.)

Fig. 4.

Full restoration LR outgrowth in PLT2-complemented plt3plt5plt7 LRP. (A–C) Confocal images of different markers in plt3plt5plt7 crossed with pPLT7_1.5::cPLT2:mRFP plt3plt5plt7 during LR outgrowth: (A) DR5::GFP, (B) pPLT2::erCFP, and (C) pSCR::H2B:YFP. Arrows indicate rescued marker expression during LR outgrowth in PLT2-complemented plt3plt5plt7. Triangles indicate cell layers in LRP at stage II/III. (D) Periclinal cell division (PeD) counts in LRP central cells at different stages and (E) emerged LR density (number per 1 cm) in WT, plt3plt5plt7, pPLT7_1.5::cPLT2:vYFP plt3plt5plt7 L-1, and pPLT7_1.5::cPLT2:vYFP plt3plt5plt7 L-2 roots at 7 d postgermination (n = 20). **P < 0.01 (Student’s t test). (F) Schematic representation of PLT-regulated auxin response, symmetry breaking markers, and tissue specificity during LR outgrowth. CEI, cortex/endodermis initial; COL, columella; Cor, cortex; Endo, endodermis; Epi, epidermis; LRC, lateral root cap; SCN, stem cell niche. (Scale bar: 100 μm.)