The Overlapping and Distinct Roles of HAM Family Genes in Arabidopsis Shoot Meristems

Abstract

In Arabidopsis shoot apical meristems (SAMs), a well-characterized regulatory loop between WUSCHEL (WUS) and CLAVATA3 (CLV3) maintains stem cell homeostasis by regulating the balance between cell proliferation and cell differentiation. WUS proteins, translated in deep cell layers, move into the overlaying stem cells to activate CLV3. The secreted peptide CLV3 then regulates WUS levels through a ligand-receptor mediated signaling cascade. CLV3 is specifically expressed in the stem cells and repressed in the deep cell layers despite presence of the WUS activator, forming an apical-basal polarity along the axis of the SAM. Previously, we proposed and validated a hypothesis that the HAIRY MERISTEM (HAM) family genes regulate this polarity, keeping the expression of CLV3 off in interior cells of the SAM. However, the specific role of each individual member of the HAM family in this process remains to be elucidated. Combining live imaging and molecular genetics, we have dissected the conserved and distinct functions of different HAM family members in control of CLV3 patterning in the SAMs and in the de novo shoot stem cell niches as well.

Keywords: Arabidopsis; HAIRY MERISTEM; confocal imaging; shoot apical meristems; shoot development; stem cells.

Figure 1

The expression of a HAM1 translational reporter in the SAM. (A–F) Confocal imaging of a pHAM1::YPET-HAM1 translational reporter in a SAM of a ham123 triple mutant, from the orthogonal view (A, D), transverse optical section view in L1 (B, E), and corpus (C, F). (A–C) merged channels from YFP (green) and PI (propidium iodide, purple). (D–F) merged channels from the quantified YFP (quantitatively indicated by color) and PI. Scale bar: 20 µm. Color bar: Fire quantification.

Figure 2

The expression of a HAM2 translational reporter in the SAM. (A–F) Confocal imaging of a pHAM2::YPET-HAM2 translational reporter in a SAM of a ham123 triple mutant, from the orthogonal view (A, D), transverse optical section view in L1 (B, E), and corpus (C, F). (A–C): merged channels from YFP (green) and PI (propidium iodide, purple). (D–F): merged channels from the quantified YFP (quantitatively indicated by color) and PI. Scale bar: 20 µm. Color bar: Fire quantification.

Figure 3

The expression of a HAM3 translational reporter in the SAM. (A–F) Confocal imaging of a pHAM3::YPET-HAM3 translational reporter in a SAM of ham123, from orthogonal view (A, D), transverse view in L1 (B, E), and corpus (C, F). (A–C): merged channels from YFP (green) and PI (purple). (D–F): merged channels from the quantified YFP (quantitatively indicated by color) and PI. Arrows indicate the boundary between shoot meristem and primordia. Scale bar: 20 µm. Color bar: Fire quantification.

Figure 4

Roles of different HAM genes in control of CLV3 patterning in SAMs. (A–F) RNA in situ hybridization of CLV3 in the SAMs of wild type (Ler) (A), ham123 (B), pHAM1::YPET-HAM1 in ham123 (C), pHAM2::YPET-HAM2 in ham123 (D), pHAM3::YPET-HAM3 in ham123 (E), and ham12 (F) grown in short days at the same developmental stage (27 DAG). Scale bar: 50 µm. At least three biological replicates were performed for each genotype with similar results.

Figure 5

The expression and function of pHAM2::YPET-HAM3 in the SAM. (A–F) Confocal imaging of a pHAM2::YPET-HAM3 translational reporter in a SAM of ham123 from orthogonal view (A, D), transverse view in L1 (B, E), and corpus (C, F). (A–C): merged channels from YFP (green) and PI (purple). (D–F): merged channels from the quantified YFP (quantitatively indicated by color) and PI. Color bar: Fire quantification. Scale bar (A–F): 20 µm. (G–I) RNA in situ hybridization of CLV3 in the SAMs of wild type (Ler) (G), ham123 (H), and pHAM2::YPET-HAM3 in ham123 (I) at the same developmental stage (27 DAG). Scale bar (G–I): 50 µm. At least three biological replicates were performed for each genotype with similar results.

Figure 6

Roles of different HAM genes in control of vegetative SAM morphology. (A–E) 3D projection views of the vegetative SAMs of indicated genotypes are shown. Ler wild type (A), ham123 (B), pHAM1::YPET-HAM1 in ham123 (C), pHAM2::YPET-HAM2 in ham123 (D), pHAM3::YPET-HAM3 in ham123 (E), and ham12 (F) were grown in the short days and imaged at the same age (28 DAG). Four biological replicates were performed for each genotype with similar results. Arrows indicate center of the SAMs. Scale bar: 20 µm.

Figure 7

Roles of different HAM genes in control of CLV3 patterning in de novo stem cell niches and in branch development. (A–E) RNA in situ hybridization of CLV3 in the initiating stem cell niches of Ler wild type (A), ham123 (B), pHAM1::YPET-HAM1 in ham123 (C), pHAM2::YPET-HAM2 in ham123 (D), and pHAM3::YPET-HAM3 in ham123 (E). Scale bar: 50 µm. Arrows indicate the CLV3 expressing cells. (F–J) Images of branches initiated from base of the cauline leaves in different genotypes, which are grown in the same condition at the same age. Arrows indicate the branches initiated normally from the base of the cauline leaves of Ler wild type (F), pHAM1::YPET-HAM1 in ham123 (H) and pHAM2::YPET-HAM2 in ham123 (I), and they indicate the absence of branches initiated from the base of the cauline leaves in ham123 (G) and pHAM3::YPET-HAM3 in ham123 (J). Scale bar: 0.5cm. At least three biological replicates were performed for each genotype with similar results.