Patterns of Stem Cell Divisions Contribute to Plant Longevity Agata Burian, Pierre Barbier de Reuille, Cris Kuhlemeier  Current Biology  Volume 26, Issue 11, Pages 1385-1394 (June 2016) DOI: 10.1016/j.cub.2016.03.067 Copyright © 2016 Elsevier Ltd Terms and Conditions

Current Biology 2016 26, 1385-1394DOI: (10.1016/j.cub.2016.03.067) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Three Models of Axillary Meristem Formation Model 1: late specification of axillary meristems. Left: the cells in the boundary region acquire leaf or internode fate and divide continuously. Middle: a subset of these cells is re-specified shortly before forming the axillary meristem. Right: cell-lineage graph: the number of cell divisions between the cells at the shoot apical meristem and axillary meristem (arrowhead) increases with internode length/leaf size. Model 2: early specification of axillary meristems; position dependence. Left: the specified cell (magenta) attains its identity as progenitor of the axillary meristem. Middle: this cell resumes division based on its position at the base of either the leaf primordium or the internode until the formation of the axillary meristem. Right: cell-lineage graph: the number of cell divisions between the cells at the shoot apical meristem and axillary meristem increases with internode length/leaf size. Model 3: early specification of axillary meristems; lineage dependence. Left: the specified cell (magenta) attains its identity as progenitor of the axillary meristem. Middle: the specified cell (magenta) does not divide until it forms the axillary meristem, whereas the surrounding cells divide continuously to form the leaf and the internode. Right: the number of cell divisions between the cells at the shoot apical meristem and axillary meristem is independent of internode length/leaf size. Asterisk indicates center of the shoot apical meristem; arrowhead indicates axillary meristem. Current Biology 2016 26, 1385-1394DOI: (10.1016/j.cub.2016.03.067) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Axillary Meristems Are Marked Early by DR5-VENUS Expression Time-lapse images of vegetative Arabidopsis shoot apex with DR5-VENUS expression. Leaf primordia are numbered from the youngest p1 to the oldest pn; i1, i2 designate progressively younger incipient primordia (i1, the stage just before bulging) as seen at T0. Dot, leaf primordium; asterisk, center of the shoot apical meristem; arrowhead, expression maximum of DR5-VENUS; black outlines, leaf primordium identified based on the curvature at T0 + 46 hr (the same cells are marked with line segment at i3 sections in (D). Results shown are representative for ten of ten leaf primordia from ten shoot apices. Scale bars, 20 μm for (A); 5 μm for (B) and (C); 10 μm for (D). See also Figure S1. (A) Top view of shoot apex. Frames specify the analyzed region of i3. (B) Curvature maps (heatmap, Gaussian curvature; crosses, directions of maximal and minimal curvature). (C) Projection of the DR5-VENUS signal from outer cell layer onto the surface. (D) Optical median longitudinal sections through i3. Current Biology 2016 26, 1385-1394DOI: (10.1016/j.cub.2016.03.067) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Ablation of the Stem Cells of the Vegetative Shoot Apex Induces the Formation of Axillary Meristems Time-lapse images of vegetative Arabidopsis shoot apices after ablation of the central zone. Images were taken every day. The image at T0, shows the apical meristem just before the ablation. White asterisk indicates the center of the shoot apical meristem; blue asterisk with corresponding dots indicates induced axillary meristem with surrounding leaf primordia; X indicates ablation. Scale bars, 20 μm. See also Figure S2. (A) Expression of CLV3-GFP. From left: top view of CLV3-GFP shoot apex, the whole shoot apex at T0 and T0 + 26 hr; for the subsequent time points, only close ups from the i2 region (framed) are shown. On the same ablated apex, axillary meristems were also induced at i4, p1, p2, and p3 (data not shown). Results shown are representative for 38/44 induced meristems from 13 ablated shoot apices. (B) Clonal analysis of the induced meristem at shoot apex with DR5-VENUS. From left: top view of projection of PI staining (gray scale) and projection of the DR5-VENUS signal (color scale) from the outer cell layer onto the surface. The whole shoot apex and close ups from i1 region (framed) at T0; for the subsequent time points, only side views of i1 region are shown. White outlines, the cell clones ultimately localized at the center of an induced axillary meristem. Results shown are representative of six out of seven induced meristems from seven ablated shoot apices. Current Biology 2016 26, 1385-1394DOI: (10.1016/j.cub.2016.03.067) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Early Sequestration of Axillary Meristem Precursors in Arabidopsis (A) Time-lapse images of Arabidopsis shoot apex starting from the vegetative phase (T0) to the early reproductive phase at T0 + 214 hr. Images were taken every 1 or 2 days; only key time points are shown. Heatmaps, the number of cell divisions for individual cells counted from T0; magenta outlines, the population of stem cells at the apical meristem at T0; black outlines, morphologically recognizable axillary meristems; white outlines, exemplary cell clones; asterisks, apical stem cells; a # at T0, the cell for which a lineage graph is shown in (B); blue, green dots at T0 + 214 hr, axillary meristem and leaf cells, respectively, from the cell-lineage graph (B), cells from abaxial leaf side are not shown; insert at T0 + 214 hr, optical median longitudinal sections through the i12 primordium with DR5-VENUS expression (arrowhead, axillary meristem). (B) Cell-lineage graph showing the fate of a single cell from the apical meristem (a # at T0) until the formation of a leaf (green dots) and axillary meristem (blue dots) at T0 + 214 hr. Dashed vertical lines, cell lineages outside the range for further imaging. (C) Number of cell divisions during cell displacement from the stem cell population of apical meristems to axillary meristems, based on the analysis of 23 axillary meristems from five apices, n = 368 cells. Scale bars, 20 μm. See also Figure S3. Current Biology 2016 26, 1385-1394DOI: (10.1016/j.cub.2016.03.067) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Early Sequestration of Axillary Meristem Precursors in Tomato (A) Time-lapse images of a tomato shoot apex during the vegetative phase. Images were taken every 2 days; only key time points are shown. Heatmaps, the number of cell divisions for individual cells counted from T0; magenta outlines, the population of stem cells at the apical meristem at T0; black outlines, morphologically recognizable axillary meristems; white outlines, exemplary cell clones; asterisks, apical stem cells; insert, optical median longitudinal sections through the shoot apex and subapical portions of the shoot where cell divisions were analyzed; arrows, the boundaries of i2 and p3 at similar developmental stages; arrowhead, axillary meristem. (B) Time-lapse images of a tomato shoot apex during the vegetative phase. Images were taken every 2 days; only the first and the last key time points are shown. White outline, a selected cell clone; asterisk, center of apical meristem; a # at T0, the cell for which a lineage graph is shown in (C); blue, red dots at T0 + 288 hr, axillary meristem and internode cells, respectively, from the cell-lineage graph (C). (C) Cell-lineage graph (from apex shown in B showing the fate of a single cell [#] adjacent to the just initiated p1 primordium at T0 until the formation of an elongated internode (red dots) and axillary meristem (blue dots) at T0 + 288 hr. (D) Number of cell divisions during cell displacement from the stem cell population of apical meristems to the boundary, based on the analysis of seven boundaries from six apices, n = 210 cells. (E) Number of cell divisions before the axillary meristem becomes apparent, counted from the boundary to the axillary meristem, based on the analysis of six axillary meristems from six apices, n = 120 cells. Scale bars, 20 μm for color maps (A) and 50 μm for sections in insert (A) and for (B). See also Figure S4. Current Biology 2016 26, 1385-1394DOI: (10.1016/j.cub.2016.03.067) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Fate of Somatic Mutations across Scales (A) The shoot apical meristem level: The stem cell population (outlined in magenta) can be divided in two subsets: the apical stem cells (dark gray, asterisks) and the surrounding subapical stem cells (light gray). Example of mutated apical (red) and subapical (blue) stem cells are shown. (B and D) The shoot level: if an apical stem cell is mutated, its descendants (red) will occupy the apical stem cells position for a long time, leading to a long clone that has a high probability to include one or more axillary meristems on a common parastichy (yellow and green arrows). If a subapical stem cell is mutated, it will be rapidly displaced from the stem cell population by the descendants of the apical stem cells, and the size of its clone (blue) will be smaller. (C and E) The tree level: a mutation occurring in an apical stem cell (red) will get fixed in at least one lateral branch, but never in all of them, increasing the genetic diversity between branches. A mutation occurring in a subapical stem cell (blue) is less likely to be fixed in the lateral branch. However, the sector will very probably occupy part of the stem cell population of the branch (e.g., more than a single cell), which increases its probability to be fixed in the next order branch. (F and G) Probability for a somatic mutation originating in either an apical or a subapical stem cell to be fixed in at least n axillary meristems (horizontal axis). When the formation of seven axillary meristems is simulated, a mutation is unlikely to become fixed in more than two of them. See also Supplemental Model Description. Current Biology 2016 26, 1385-1394DOI: (10.1016/j.cub.2016.03.067) Copyright © 2016 Elsevier Ltd Terms and Conditions