Patterns of Stem Cell Divisions Contribute to Plant Longevity

Article

Patterns of Stem Cell Divisions Contribute to Plant Longevity Graphical Abstract

Authors Agata Burian, Pierre Barbier de Reuille, Cris Kuhlemeier

Correspondence [email protected]

In Brief Trees can live for thousands of years despite aging-related accumulation of somatic mutations. Burian et al. use quantitative cell-lineage analysis and computational modeling to show that early sequestration of plant stem cells and reduction of divisions in the stem cell lineage prevent fixation of somatic mutations, slowing down mutational meltdown.

Highlights d

Axillary meristems are set aside early, analogous to the metazoan germline

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Only 7–9 cell divisions separate branching events, independent of branch length

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Patterns of stem cell divisions promote genetic heterogeneity

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Slowdown of mutational meltdown is the basis for tree longevity

Burian et al., 2016, Current Biology 26, 1–10 June 6, 2016 ª 2016 Elsevier Ltd. http://dx.doi.org/10.1016/j.cub.2016.03.067

Please cite this article in press as: Burian et al., Patterns of Stem Cell Divisions Contribute to Plant Longevity, Current Biology (2016), http://dx.doi.org/ 10.1016/j.cub.2016.03.067

Current Biology

Article Patterns of Stem Cell Divisions Contribute to Plant Longevity Agata Burian,1,2 Pierre Barbier de Reuille,1 and Cris Kuhlemeier1,* 1Institute

of Plant Sciences, University of Bern, 3013 Bern, Switzerland of Biophysics and Morphogenesis of Plants, University of Silesia in Katowice, 40-032 Katowice, Poland *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2016.03.067 2Department

SUMMARY

The lifespan of plants ranges from a few weeks in annuals to thousands of years in trees. It is hard to explain such extreme longevity considering that DNA replication errors inevitably cause mutations. Without purging through meiotic recombination, the accumulation of somatic mutations will eventually result in mutational meltdown, a phenomenon known as Muller’s ratchet. Nevertheless, the lifespan of trees is limited more often by incidental disease or structural damage than by genetic aging. The key determinants of tree architecture are the axillary meristems, which form in the axils of leaves and grow out to form branches. The number of branches is low in annual plants, but in perennial plants iterative branching can result in thousands of terminal branches. Here, we use stem cell ablation and quantitative cell-lineage analysis to show that axillary meristems are set aside early, analogous to the metazoan germline. While neighboring cells divide vigorously, axillary meristem precursors maintain a quiescent state, with only 7–9 cell divisions occurring between the apical and axillary meristem. During iterative branching, the number of branches increases exponentially, while the number of cell divisions increases linearly. Moreover, computational modeling shows that stem cell arrangement and positioning of axillary meristems distribute somatic mutations around the main shoot, preventing their fixation and maximizing genetic heterogeneity. These features slow down Muller’s ratchet and thereby extend lifespan. INTRODUCTION When a plant emerges from the seed, it is a simple structure: root, hypocotyl, cotyledons, and a shoot apical meristem with a few true leaves. All further above-ground cells, tissues, and organs arise post-germination as the descendants of the stem cells at the tip of the shoot apical meristem. The lifespan of a plant can range from a few weeks in annuals, to hundreds of years in some slow-growing herbs, to thousands or even tens of thousands of years in trees [1–4]. While the relationships between growth rate, aging, mortality, and reproduction are

complex and differ among plants and animals [5], the extreme longevity of large trees poses a particularly interesting conundrum. DNA replication errors are a main source of mutations, and indeterminate growth will cause the accumulation of somatic mutations with each cell cycle. Without purging through recombination, the accumulation of deleterious mutations will eventually cause mutational meltdown, a phenomenon known as Muller’s ratchet [6–8]. The key determinants of tree architecture are the axillary meristems, which form in the axils of leaves. They develop into axillary buds, which can grow out and form branches in response to developmental and environmental signals [9–12]. During bud outgrowth, the axillary meristem becomes the apical meristem of the branch. In the leaf axils of the branch, secondary axillary meristems form that can grow out into secondary branches. The number of branches is low in annual plants, but in perennial plants iterative branching can result in thousands of terminal branches. Thus, branching is comparable between annuals and perennials, the main difference being the number of iterations. Knowing the number of cell divisions separating the embryonic meristem from the terminal branches is crucial to determine the rate of genetic aging. This number is the number of cell divisions between an apical and an axillary meristem multiplied by the number of iterations. The number of iterations can be estimated with reasonable accuracy. For example, assuming regular bifurcating branching, ten iterations will give 1,024 (210) terminal branches. In reality, branching will be less regular, as it will depend on environmental, physiological, and genetic factors. However, in principle, the exact topology of the branching system can be determined in any individual tree [13–15]. The number of cell divisions between apical and axillary meristems is not known. Clonal analyses have shown that somatic sectors can extend through multiple nodes and encompass leaves, axillary meristems, and internodes [16–19]. This proves that a single cell can contribute to multiple organs, but it is hard to extrapolate back from the sector to its founder cell. We consider three possible models (Figure 1). In model 1, axillary meristems derive from differentiated shoot cells, either of the internode or the leaf (late specification of axillary meristem) [20, 21]. These differentiated cells undergo multiple cell divisions during elongation of the internode or outgrowth of the leaf. In this model, the number of cell divisions between the apical meristem and the terminal organs correlates with plant stature and will become very high in big-statured plants, as favored by population geneticists [22–24]. Current Biology 26, 1–10, June 6, 2016 ª 2016 Elsevier Ltd. 1

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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.

Models 2 and 3 take into account that cell division ceases during the formation of the boundary between the leaf primordium and the apical meristem (the position of the future leaf axil) [10, 25] and that gene expression studies indicate the separate identity of the boundary [26–29]. Note, however, that the formation of an axillary meristem is delayed relative to the formation of the subtending leaf. In Arabidopsis, this delay can be as much as 11 plastochrons, that is, the first anatomical indication of axillary meristem formation occurs in the axil of a leaf that has already ten younger leaves above it on the stem [30]. The patterns of cell division during those ten plastochrons are not known. In model 2, we assume that cells in the leaf axil engage in cell division until a few of them develop into an axillary meristem, and that axillary meristem specification is position dependent. If this is correct, the number of cell divisions between the 2 Current Biology 26, 1–10, June 6, 2016

apical meristem and the terminal organs correlates with plant stature, as in model 1. Model 3 assumes that cells in the leaf axil retain their quiescent state until axillary meristems are formed, and that axillary meristem specification is lineage dependent. In this model, the number of cell divisions between apical and axillary meristems will be low and not related to plant stature. Therefore, key questions to be answered are whether axillary meristems are specified at the same time as the subtending leaf, and whether progenitor cells of axillary meristems maintain a quiescent state. Unless a mutation arises in the zygote, the resulting individual will consist of sectors with different genotypes. Indeed, chimerism appears to be common in plants [31–33] and especially long-lived trees should be considered not as single organisms but as colonies of competing branches [34–36]. For the case of selectively neutral mutations, the extent of mutant sectors depends on the number and pattern of cell divisions. The largest sectors derive from mutations occurring in stem cells. How does a mutation that arises in a stem cell of the shoot apical meristem propagate through the plant and what is the chance of fixation? The width of the widest clonal sectors suggests that one to three stem cells contribute to the circumference of the stem. Their length suggests that stem cells are not permanent, with interpretations ranging from essentially non-permanent (‘‘temporary inhabitants of a permanent office’’ [37–39]) to surprisingly stable [40]. Existing mathematical models, which are based on the assumption that stem cells in the shoot apical meristem are non-permanent, conclude that the chance of fixation of somatic mutations is high [41, 42]. Here, we use surgical manipulations and quantitative cell-lineage analysis in Arabidopsis and tomato to show that axillary meristem specification occurs at the shoot apical meristem concomitant with leaf initiation, and that the number of divisions in the cell lineage from the shoot apical meristem to the axillary meristem is reduced in comparison with the cell lineage leading to differentiated cells. We use the quantitative data to generate a computational model that recreates the patterns of cell division from the stem cells in the shoot apical meristem to the stem cells in the axillary meristems. The realistically modeled cell lineages indicate that stem cells persist at the shoot apex. This stem cell behavior prevents fixation and instead maximizes genetic heterogeneity, which will become more pronounced with increasing age. We extrapolate our data from Arabidopsis and tomato to long-lived trees. The tree growth habit has evolved in numerous plant lineages [43]; thus, notwithstanding differences in important developmental details, early sequestration of axillary meristems and semi-permanence of apical stem cells are conserved features of seed plants. RESULTS AND DISCUSSION Axillary Meristems Are Specified Early If axillary meristems are specified early, their progenitor cells should be marked by specific gene expression. To determine the origin of the cells incorporated into the axillary meristem, we performed time-lapse imaging in vegetative Arabidopsis apices expressing the auxin response reporter DR5-VENUS. Leaf primordia are numbered from the youngest, p1, to the oldest, pn, whereas i1, i2, i3 etc. designate incipient primordia, with

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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 mm for (A); 5 mm for (B) and (C); 10 mm 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.

i1 being the oldest stage just before bulging. DR5-VENUS is not expressed at the i3 stage (T0, Figures 2A–2D), but expression is apparent 23 hr later in the future boundary (leaf axil) and is maintained at the boundary during primordium bulging (T0 + 23 hr, T0 + 46 hr, Figures 2A–2D). Thus, even though axillary meristems develop as much as 11 plastochrons after the subtending leaves [30], DR5-VENUS expression marks the position of the future axillary meristem and not the position of the leaf. This applies to the vegetative phase but also after the transition to flowering, when the axillary meristems produce the inflorescence or flowers and the leaf is reduced to a (cryptic) bract (Figures S1A–S1C). If axillary meristems are specified early, their further development must be inhibited. It has been hypothesized that the center of the vegetative meristem is a source of an inhibitor of the formation of axillary meristems [44]. To test this hypothesis, we ablated the central zone and followed the development of ablated apices by time-lapse imaging. Ablation eliminated expression of the stem cell marker CLV3-GFP (Figure 3A). However, development of existing and incipient leaf primordia continued, showing that ablation does not inhibit growth per se (e.g., i2 in Figure 3A; i1–i3 in Figure S2). After ablation, expression of CLV3-GFP marked the induction of stem cells that developed into meristems (Figure 3A). Clonal analysis showed that the cells of such induced meristems were recruited from the region expressing DR5-VENUS adjacent to the incipient leaf primor-

dium (Figure 3B). Thus, these meristems can be considered as prematurely initiated axillary meristems. In summary, we conclude that in the intact vegetative shoot apex the formation of axillary meristems is delayed due to inhibitory effect from the central zone, whereas the specification is an early event that occurs at the shoot apical meristem concomitant with leaf initiation. This is inconsistent with model 1 (Figure 1). Number of Cell Divisions in Cell Lineage Leading to the Axillary Meristem Is Reduced in Comparison with Differentiated Cells The exact cell lineages leading to the formation of axillary meristems are not known, and several scenarios with different outcomes are possible (Figure 1). In order to determine the patterns and numbers of cell division between the apical and axillary meristem, we performed cell-lineage analysis in both Arabidopsis and tomato. Note that all the data presented here concern the protodermal (L1) layer, but that it is likely that our conclusions will also be valid for the internal layers. Unlike Arabidopsis, which forms a rosette of leaves, axillary meristems in tomato are separated from the apical meristem by elongated internodes. If axillary meristems derive from quiescent cells, then the number of cell divisions in the cell lineage leading to axillary meristems in tomato should be similar to Arabidopsis. However, if axillary meristems derive from repeatedly dividing cells of an internode or a leaf, this number should be increased in tomato. In Arabidopsis, we analyzed cell lineages at shoot apices starting from the vegetative phase until the early reproductive phase. First, we identified a population of 13–15 stem cells at T0 as a reference for our analysis (outlined in magenta in Figure 4A). This number is similar to the number of cells expressing CLV3 Current Biology 26, 1–10, June 6, 2016 3

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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 mm. 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 DR5VENUS 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.

(14–20 cells are marked by CLV3-GFP, n = 13 apical meristems). Within this stem cell population, we recognized a subset of three to four cells, that we name apical stem cells (asterisks at T0, Figure 4A). These cells retain their position at the meristem center during prolonged live imaging and thus are (semi-)permanent, an attribute that has important consequences, as will be discussed below. The apical stem cells slowly divide to give rise to an apical stem cell and a subapical stem cell; the latter further divides to provide precursors for new organs (Figure 4A). During early reproductive phase, Arabidopsis axillary meristems become morphologically recognizable at the boundary of leaf primordia, up to 60 mm from the apical meristem center (T0 + 171 hr, T0 + 214 hr, Figure 4A). The number of cell divisions counting from the population of stem cells at the apical meristem at T0 to the axillary meristems is around four to eight (i12–i14 at T0 + 214 hr, Figures 4A and 4C; Figure S3). For comparison, the number of divisions in cells that formed leaf primordia is usually higher (Figures 4A and 4B). In tomato, axillary meristems are morphologically recognizable approximately 300–550 mm from the apical meristem center, that is several times more than in Arabidopsis (Figures 5A and 5B). Due to this large distance, it was technically difficult to continuously follow cell fates from the apical to the axillary 4 Current Biology 26, 1–10, June 6, 2016

meristem in tomato. Thus, we analyzed cell lineages in two steps: (1) from the population of stem cells at the apical meristem to the boundary of a leaf primordium; and (2) from a different leaf boundary at similar developmental stage to the axillary meristem (e.g., the boundary of i2 at T0 + 192 hr is of comparable developmental stage as the boundary of p3 at T0, arrows, Figure 5A). As in Arabidopsis, we identified in tomato a population of stem cells (outlined in magenta at T0, Figure 5A) with a subset of three to four (rarely five) semi-permanent apical stem cells. Similar numbers of apical stem cells have been found also in other species [38, 40, 45]. The number of cell divisions counting from the stem cell population to the boundary is four to eight (boundary of i2 at T0 + 192 hr, Figures 5A and 5D; Figures S4A, S4B, and S4F). Next, from the boundary to the axillary meristem there occur one to three cell divisions (boundary of p3 at T0 + 144 hr, Figures 5A and 5E; Figures S4C, S4D, S4G, and S4H). Further cell divisions increase the size of the axillary meristem (T0 + 192 hr, Figure 5A). During this stage, the frequency of cell divisions in the internode is much higher (at T0 + 192 hr, Figure 5A, six to eight divisions in the internode and two to three divisions in the axillary meristem; another example is shown in Figures 5B and 5C). This higher mitotic activity in the internode together with an intensive cell elongation accounts for the increase in the distance between

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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 mm. See also Figure S3.

the apical meristem and the axillary meristem. Thus, most cell divisions involved in axillary meristem formation occur between the center and the boundary of the apical meristem. As soon as a cell reaches the boundary, the mitotic activity is reduced, and only few cell divisions are needed to establish a functional axillary meristem. Therefore, our results support model 3 (Figure 1) and are in agreement with previous studies in maize showing that cells of the apical meristem undergo a progressive restriction of cell developmental commitment [46]. The early specification and low rate of subsequent cell division are reminiscent of the germline in metazoans, and we propose that, similarly, the cells of the axillary meristems are set aside early reducing the accumulation of replicative errors. The conservation of the mechanisms of axillary meristem development makes it possible to extrapolate our quantitative data from annual plants to trees [47, 48]. Making this extrapolation, ten branching events from the embryonic shoot apical meristem

will give rise to 1,024 (210) terminal meristems, which, according to the data presented here, will each have undergone about 60 cell divisions. This number only doubles in a tree with a million branches. In comparison, an estimated 30–50 cell divisions per generation occur in annuals such Arabidopsis or maize [49, 50]. Patterns of Stem Cell Divisions Maximize Genetic Heterogeneity If apical stem cells were strictly non-permanent, as was assumed in earlier mathematical models [41, 42], the chance of fixation of a neutral mutation is 1/n where n is the number of stem cells, that is 33%, assuming three stem cells. Because the apical stem cells are instead semi-permanent (Figures 4 and 5; Figures S3 and S4), the chance of fixation will decrease. We tested the magnitude of the effect using computational modeling. We constructed a model of a growing plant tissue that quantitatively reproduces key characteristics, such as cell-size distribution, average Current Biology 26, 1–10, June 6, 2016 5

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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 mm for color maps (A) and 50 mm for sections in insert (A) and for (B). See also Figure S4.

cell-division time, and distribution of the number of cell divisions from the apical meristem to the axillary meristem (Figure 6; for model description, see Supplemental Model Description). We used the model to calculate the chance of a neutral mutation to be fixed in either the main shoot or in an axillary meristem. The 6 Current Biology 26, 1–10, June 6, 2016

probability of fixation within the main shoot was approximately 0.15% for an apical stem cell mutation and essentially zero for subapical stem cells. Thus, the chance of fixation of a mutation within the main shoot is 20 times lower than previously calculated [34, 42] and in line with assumptions based on clonal analyses

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[51]. It is important to note that a mutation that arises in an apical stem cell will propagate as a long sector with a width of one-third to one-fourth of the circumference of the main shoot, whereas a mutation in a subapical stem cell will be rapidly displaced from the meristem and affect only a narrow and short section of the shoot (Figures 6A, 6B, and 6D). Similar geometrical considerations apply to the chance of a mutation to become fixed in axillary meristems. The chance of fixation in all axillary meristems is essentially zero. However, the chance of fixation in at least one axillary meristem is 61% when a mutation occurred in an apical stem cell and 38% when it occurred in a subapical stem cell (Figures 6F and 6G; see Supplemental Model Description). When the axillary meristem grows out, it becomes the apical meristem of the branch. Even when new mutations in this branch meristem are not fixed, they have a high chance of fixation in at least one second order axillary meristem, and so on in an iterative process (Figures 6C and 6E). For non-neutral mutations, this will result in competition between branches as predicted by computational models of tree growth and consistent with observations [35, 52]. In either case, a tree will contain nested sets of mutations. The model predicts that the vast majority of somatic mutations will be present in small sectors and that most of them will not propagate into the gametes. This is equally true for non-replicative mutations, except that, e.g., environmentally induced mutations will accumulate, irrespective of whether cells divide or not. This difference also applies in short-lived annual plants and may explain the relative scarcity of replication-based mutations in Arabidopsis [53]. When axillary buds are dormant for a prolonged amount of time, as often happens in trees, the proportion of replicative mutations will decrease. This provides a mechanistic basis for phylogenetic studies that estimate the rate of molecular evolution in relation to generation time [54, 55]. With time, somatic mutations will inevitably accumulate and cause aging-related defects [56, 57]. In humans, accumulation of somatic mutations with age is the primary cause of cancer [58–61]. This is unlikely to be due to more efficient DNA repair. DNA repair pathways are conserved between plants and animals and plant stem cells are highly susceptible to DNA damage [62]. Yet, plant lifespan is limited more often by structural damage and incidental disease [1]. We propose that stem cell sequestration, meristem geometry, and intra-organismal competition delay mutational meltdown and thereby extend the lifespan of plants. EXPERIMENTAL PROCEDURES

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

Plant Material and Growth Conditions Arabidopsis plants were grown under short-day conditions (8 hr of light, 65% humidity, 22 C [light], and 18 C [dark] temperature, irradiance of 150 mE m–2 s–1) to prolong the vegetative phase of development. Plants were germinated and

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.

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Please cite this article in press as: Burian et al., Patterns of Stem Cell Divisions Contribute to Plant Longevity, Current Biology (2016), http://dx.doi.org/ 10.1016/j.cub.2016.03.067

grown in soil. For in vivo imaging of vegetative shoot apices, 30- to 45-day-old rosettes were used; for imaging of the shoot apices after the transition to flowering (reproductive phase) at the earlier and later stage, 46- to 66- and 67- to 77day-old rosettes were used, respectively. The following transgenic lines of Arabidopsis were used: DR5::VENUS-N7 in Columbia background [63]; CLV3::mGFP5-ER in heterozygous pin1–7 background (Landsberg ecotype) [64]; and TCS::GFP [65]. To make the shoot apex accessible for observation in the confocal microscope, the rosettes were dissected using a stereo-microscope (Nikon SM2 1500). All leaves and leaf primordia that covered the apical meristem were cut off, leaving only the youngest primordia (p1–p6), and older leaves that do not cover the meristem. The dissected rosettes were grown in Petri dishes filled with Murashige-Skoog (MS) medium (pH 5.8 adjusted with KOH) containing 2% sucrose, 1.5% agarose, 0.1 mM gibberellic acid A3 (Sigma-Aldrich), and 0.1 mM kinetin (Sigma-Aldrich) supplemented with 1 mL/ml PPM (Plant Cell Technology) as a preservative for tissue culture. For long-term in vitro culturing (time lapses 72 hr and longer; Figures 3, 4, and 5; Figures S2, S3, and S4), cytokinins (zeatin [mixed isomers, Sigma-Aldrich] or BAP [Sigma-Aldrich] alone, or combined with IAA [Sigma-Aldrich]) were added (5 ml of 1 mM hormone solution per day). Arabidopsis shoot apices in such in vitro culture continued to grow and produce new primordia according to the normal spiral phyllotactic pattern. Leaf primordia that grew out during the time lapse and covered the apical meristem were trimmed before each imaging. Tomato plants (Lycopersicum esculentum cv. Moneymaker) were grown under long-day conditions (16 hr of light, 65% humidity, 22 C [light] and 18 C [dark] temperature, irradiance of 110 mE m–2 s–1). Transgenic line pDR5rev:3xVENUS-N7 was used [66]. Plants were germinated and grown in soil. For in vivo imaging of vegetative shoot apices, 7-day-old seedlings were used. The shoot apices were dissected, leaving only the youngest leaf primordia (p1–p2) and transferred into the MS medium (the same as for Arabidopsis). The base of the shoot was trimmed, and apices were transferred to new MS medium every 6 days. New outgrowing leaf primordia were trimmed before imaging in the microscope. Ablation Experiments Ablation of the central zone at the Arabidopsis vegetative shoot apices was performed manually with solid tungsten shafts with point radius of 0.5 mm (Picoprobe, GGB Industries). The ablated shoot apices were imaged in the confocal microscope with a time interval of about 24 or 48 hr, which was followed by hormone treatment to maintain growth of the apex in long-term time lapse. Hormone treatment of intact shoot apices did not induce axillary meristems within the apex (based on analysis of 12 apices). In ablation experiments, we used three lines: DR5::VENUS-N7, CLV3::mGFP5-ER, and TCS::GFP. All these lines gave similar results with regard to the induction of axillary meristems after the ablation. Confocal Microscopy The shoot apices were imaged with confocal microscopy at one time point or through several time points in time-lapse sequence with time intervals of about 12, 24, or 48 hr. Between microscopic observations, the plants were kept either in short-day (Arabidopsis) or long-day (tomato) conditions. Microscopic observations were carried out using an upright confocal laserscanning microscope (Leica TCS SP5) with long-working distance water immersion objectives 633 or 203. The cell wall was stained with 0.1% propidium iodide (PI; Sigma-Aldrich) for 3–10 min. For imaging, shoot apices growing in a Petri dish were covered with water. To reduce medium swelling due to water absorption during the scanning in the microscope, water was added to the Petri dish 10–15 min before imaging. Excitation was at 488 nm at 20% full power (and 30% laser output). The image collection was at 505–545 nm for VENUS, 500–540 nm for GFP, and 600–656 nm for propidium iodide. Scanning speed was set at 400 Hz with 512 3 512-pixel frames. The pinhole was set at 1AE, 13 zoom. The Z intervals of sections in stacks were 0.08–0.21 mm for Arabidopsis and 0.5–1 mm for tomato. Images were collected at 12 bits. Image Processing and Analysis Original confocal z-stack images were processed in Fiji (http://fiji.sc/) to obtain .tiff files, which were further processed and analyzed with MorphoGraphX (MGX) software [67].

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The stacks with propidium iodide (PI) channel (cell-wall visualization) were first cleaned from the noise with 3D filters. The top views and optical longitudinal median sections through the primordia (e.g., Figures 2A, 2D, 3A, and S1) (sections of 2–3 mm thickness) were made by combing PI channel with unprocessed GFP or VENUS channel. To quantify the fluorescent signal (e.g., Figures 2C and 3B), the signal was projected from the defined depth onto the surface (i.e., 1–6 mm, which accounted for the outermost meristem layer–L1). The tissue geometry was quantified by computing the curvature tensor of its surface, from which we extract the Gaussian curvature, as described [67]. Cell-Lineage Analysis For analysis of cell fates and cell-division numbers in time-lapse series, confocal z stacks with the PI channel were processed in MorphoGraphX to create 2D projections of the cell outlines. 2D images were next analyzed in Point Tracker software [68] to generate heatmaps of the number of cell divisions for individual cells. Point Tracker enables to trace cell fates by manual identification of cells at consecutive time points based on their junctions (cell vertices) and saving information about sequences of divisions in individual cells, which, in turn, is transformed into heatmaps. To estimate the number of divisions from the stem cells of the apical meristem to the axillary meristem (in Arabidopsis, Figure 4C) or to the boundary (in tomato, Figures 5D and 5E), we considered the cell clones that originated from one stem cell in the population identified at T0, or from the cells located close to that population (Figures 4A and 5A; Figures S3B and S4B). In the latter, the ultimate number of cell divisions for individual cells (read from the heatmaps) for such a clone needs to be increased by the presumable number of cell divisions that took place from the moment that they left the population of stem cells. This was made by the identification of cell complexes at T0 representing small cell clones and by deduction of the cell division sequence based on different cell-wall thickness (Figures S3C and S4E). Cell-lineage graphs were prepared by manually following individual cells in 2D images showing cell outlines. Histogram plots were performed with Origin (OriginLab). SUPPLEMENTAL INFORMATION Supplemental Information includes four figures and Supplemental Model Description and can be found with this article online at http://dx.doi.org/10. 1016/j.cub.2016.03.067. AUTHOR CONTRIBUTIONS A.B. performed the experiments. P.B.d.R. performed the computational modeling. A.B, P.B.d.R., and C.K. conceived the project and wrote the manuscript. ACKNOWLEDGMENTS We thank T. Vernoux, N. Ori, and B. Mu¨ller for sharing materials and D. Kwiatkowska, F. Hartmann, and S. Robinson for helpful comments. This work was supported by grants from the Swiss National Science Foundation and SystemsX.ch, the Swiss Initiative in Systems Biology. Received: February 25, 2016 Revised: March 23, 2016 Accepted: March 29, 2016 Published: May 5, 2016 REFERENCES 1. Lanner, R.M. (2002). Why do trees live so long? Ageing Res. Rev. 1, 653–671. 2. Lynch, A.J.J., Barnes, R.W., Cambecedes, J., and Vaillancourt, R.E. (1998). Genetic evidence that Lomatia tasmanica (Proteaceae) is an ancient clone. Aust. J. Bot. 46, 25–33.

Please cite this article in press as: Burian et al., Patterns of Stem Cell Divisions Contribute to Plant Longevity, Current Biology (2016), http://dx.doi.org/ 10.1016/j.cub.2016.03.067

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