Cyclic programmed cell death stimulates hormone signaling and root development in Arabidopsis

Cell death establishes a site for development As plant roots grow through the soil, lateral roots emerge to reach more resources. Xuan et al. now show that programmed cell death sets the course for lateral root development. Cells in a specialized region of the root cap periodically die off as a group, defining a location at which a lateral root will later develop. Science, this issue p. 384 Cycles of programmed cell death establish the developmental clock in plant roots. The plant root cap, surrounding the very tip of the growing root, perceives and transmits environmental signals to the inner root tissues. In Arabidopsis thaliana, auxin released by the root cap contributes to the regular spacing of lateral organs along the primary root axis. Here, we show that the periodicity of lateral organ induction is driven by recurrent programmed cell death at the most distal edge of the root cap. We suggest that synchronous bursts of cell death in lateral root cap cells release pulses of auxin to surrounding root tissues, establishing the pattern for lateral root formation. The dynamics of root cap turnover may therefore coordinate primary root growth with root branching in order to optimize the uptake of water and nutrients from the soil.

. ACKNOWLEDGMENTS We thank D. Rowitch (UCSF) for Olig2-GFP mice and comments on the manuscript. Procurements of human brain tissues from the UCSF Pediatric Neuropathology Research Laboratory have been supported by grants from the University of California (142657), the National Institute of Neurological Disorders and Stroke (1P01 NS083513), and HHMI. J.C. is supported by an American Heart Association Postdoctoral Fellowship (15POST23020039). This work was supported by grants from the NIH (1R01NS064517 to C.J.K.) and the National Multiple Sclerosis Society (RG 5216-A-1 to S.P.J.F.) and Race to Erase MS (to S.P.J.F.). The supplementary materials contain additional data. SUPPLEMENTARY  The plant root cap, surrounding the very tip of the growing root, perceives and transmits environmental signals to the inner root tissues. In Arabidopsis thaliana, auxin released by the root cap contributes to the regular spacing of lateral organs along the primary root axis. Here, we show that the periodicity of lateral organ induction is driven by recurrent programmed cell death at the most distal edge of the root cap. We suggest that synchronous bursts of cell death in lateral root cap cells release pulses of auxin to surrounding root tissues, establishing the pattern for lateral root formation. The dynamics of root cap turnover may therefore coordinate primary root growth with root branching in order to optimize the uptake of water and nutrients from the soil.
T he root cap is the outermost tissue covering the root tip and represents a major rootrhizosphere interaction site (1-3). It is commonly recognized as a protective tissue for the meristematic cells of the root apex and as a sensory organ that perceives en-vironmental signals such as gravity, water, and nutrients to direct root growth (4)(5)(6). Although it persists during the life span of roots, it is subjected to a regeneration process in which new cell layers are continuously produced internally while superficial cell layers are regularly sloughed off. In Arabidopsis, the root cap consists of a central columella and peripheral lateral root cap cells (7). Programmed cell death (PCD) of lateral root cap cells occurs when they reach the onset of the elongation zone (8,9) S1A). This region is also designated as the oscillation zone because it displays massive oscillations in gene expression (10). These oscillations periodically define the prebranch sites, which may further develop as lateral roots (10). Root cap-specific conversion of the auxin precursor indole-3-butyric acid (IBA) into indole-3-acetic acid (IAA) creates a local auxin source that is essential for the oscillating transcriptional mechanism, which installs the regular spacing of lateral roots (11,12).
Analysis of the transcriptional auxin signaling output reporter DR5rev:VENUS-N7 (13) by means of stereomicroscopy revealed a striped DR5 pattern in the most distal lateral root cap cells, a pattern that could also be observed for the root cap-expressed early-stage PCD marker pPASPA>>H2A-GFP (GFP, green fluorescent protein) (Fig. 1, A and B, and fig. S1) (8). In vivo timelapse imaging of vertically growing roots showed that the most distal stripe of DR5 expression faded out every~4 hours ( Fig. 1C; fig. S2, A and B; and movie S1). When tracing back the site of origin of lateral root primordia (n = 96 primordia) (Fig. 1C), we found that all primordia were initiated at positions where a distal DR5 stripe had vanished. Furthermore, the disappearance of the DR5 signal from the lateral root cap preceeded the DR5:Luciferase maximum in the oscillation zone ( fig. S3) and occurred with a similar periodicity ( fig. S2B). By rotating the roots by 135°, the orientation of root growth is corrected toward the gravity vector, and a bend is formed. During the reorientation, the period of DR5 oscillations in the oscillation zone is transiently shortened, and lateral root formation is stimulated (10,(14)(15)(16). Likewise, the period between successive losses of DR5 stripes was also shortened from~4 to~2 hours (fig. S2, C and D, and movie S2). Altogether, these results show that the disappearance of the DR5 signal from the lateral root cap, the DR5 oscillations in the oscillation zone, and the formation of lateral root primordium are temporally and spatially interconnected.
The longitudinal extent of the lateral root cap is developmentally restricted by induction of PCD in the most distal lateral root cap cells (8), raising the possibility that the periodic disappearance of the DR5 signal coincides with PCD in the lateral root cap. Consistently, pPASPA3>>H2A-GFP showed a striped pattern in the lateral root cap (Fig. 1B). Moreover, coexpression of the DR5rev: VENUS-N7 reporter with pPASPA3:NLS-tdTomato revealed overlapping expression in the most distal lateral root cap cells ( Fig. 2A and fig. S4A). Timelapses showed that both signals disappeared synchronously ( Fig. 2A and fig. S4B), with a period of~4 hours ( fig. S2B) and spatially correlating with sites of new lateral root primordia (movie S3). Moreover, a 135°gravistimulation also transiently decreased the periodicity of disappearance of PASPA3 stripes to~2 hours ( fig. S2D and movie S4). Thus, PCD in the lateral root cap is predictive of lateral root formation.
In Arabidopsis, the accurate timing of PCD in the lateral root cap requires the transcription factor SOMBRERO (SMB) (8,17). pSMB:NLS-GFP stripes overlapped with pPASPA3:NLS-tdTomato stripes in the most distal lateral root cap and disappeared every~4 hours (figs. S2B and S4C). The smb-3 mutant exhibits delayed PCD of the lateral root cap cells (8,17) and as a result has an increased number of the lateral root cap cells that ectopically extend into the elongation zone ( fig. S5, A and B) (8,17). In this mutant, the typical stripe-like pattern of DR5 expression had disappeared (Fig. 2B) S6B). Additionally, these roots lacked DR5 stripes (Fig. 2C) and DR5:Luciferase oscillations (Fig. 2, D and E, and movie S5), and the numbers of prebranch sites and lateral roots were reduced, respectively, by 79.4 and 87.5% at 0.3 mM Dex (Fig. 2F and fig. S6, A and C). When plants were transferred back to control medium, the newly formed root segment reestablished normal growth with the production of a normal lateral root cap and lateral roots (fig. S6, D to F). In contrast, the part of the root that was formed during Dex treatment remained devoid of lateral roots ( fig. S6, D and E). These results indicate that the controlled and recurrent PCD of the lateral root cap cells is the driving factor for gene expression oscillations in the oscillation zone and subsequent lateral root induction.
Oscillations are modulated by a local auxin source in the root cap, derived from the auxin precursor IBA (11,12). Moreover, genetic ablation of the lateral root cap cells repressed the capacity to produce extra lateral roots in response to exogenous IBA application in Dextreated 35S:SMB-GR ( fig. S6G). Therefore, we asked whether the auxin response that we observed in the root cap itself could be required for lateral root patterning. We conditionally repressed the auxin response in the lateral root cap cells through activation of a stabilized allele of the auxin response repressor IAA17/ AXR3 (pSMB:axr3-1-GR) (5,18). Dex treatment resulted in agravitropic root growth ( fig. S7A) and loss of DR5 expression in the lateral root cap cells (fig. S7, B and C), but this did not alter the PCD process ( fig. S7D) and did not affect the lateral root number (fig. S7E). Constitutive transactivation of UAS:axr3-1 in the lateral root cap only slightly reduced lateral root formation, whereas transactivation of UAS:axr3-1 in xylem pole pericycle cells blocked lateral root formation ( fig. S7F) (19). Therefore, a transcriptional auxin response in the lateral root cap itself is not a decisive factor for lateral root patterning.  Alternatively, auxin transport from the root cap to the root proper could be the connecting element for the oscillatory behavior in gene expression in the elongation zone. Consistently, timelapse analyses of the semiquantitative auxin input reporter R2D2 (20) revealed a marked increase of auxin levels in epidermal cells, before loss of cellular integrity of adjacent lateral root cap cells (Fig. 3A and movie S6). This suggests that auxin released from lateral root cap cells during a late stage of PCD is efficiently taken up by the abutting epidermal cells. To understand how this could result in auxin signaling in stele cells of the oscillation zone, we adopted an in silico auxin-transport model (21) to simulate the auxin dynamics in the root apex (further details are available in the supplementary materials). Simulating the PCD of distal lateral root cap cells, under the assumption that PCD leads to a release of auxin into the surrounding apoplast, generated a transient auxin peak in stele cells in the elongation zone (Fig. 3, B to D; fig. S8; and movie S7), which is consistent with the oscillating activation of the DR5:Luciferase. When defects in either auxin uptake or IBA conversion are prescribed, the model fails to predict such a transient increase in stele auxin levels after lateral root cap cell turnover (Fig. 3 S9, A and B), whereas auxin production was predicted to create high auxin levels in the lateral root cap before PCD.
Consistent with these simulations, we observed a reduced DR5 signal in the lateral root cap that was correlated with less prebranch sites and lateral roots in ibr1ibr3ibr10 and aux1 mutants (Fig. 4, A and B). Moreover, PCD in the most distal lateral root cap cells was closely associated with increased auxin in the underlying epidermal cells ( Fig. 3A and movie S6). We further tested the contribution of auxin transport within the lateral root cap by means of tissue-specific complementation of the aux1 mutant. In agreement with model predictions ( fig. S9, C to E), transactivation of AUX1 in the root cap rescued the defect in lateral root formation and agravitropic growth of aux1 mutants ( fig. S9, F and G). Thus, auxin transported within the (lateral) root cap allows the root cap to communicate with the elongation zone for establishing sites for lateral roots to develop. This process ensures that IBA-derived auxin can be transported toward the oscillation zone.
The auxin-transport topologies in our model also include carrier-mediated efflux and apoplastic diffusion. In the presence of influx carriers and auxin production, simulations lacking carrier-mediated efflux failed to generate an auxin transient in the elongation zone but generated an auxin accumulation in the lateral root cap (Fig. 3, B to D; fig. S8; and movie S7). In the model, diffusion rates were positively correlated with the strength of the auxin peak in the stele. However, apoplastic diffusion could not compensate for a lack in auxin efflux in our simulations ( fig. S10). In an attempt to identify the components of this auxin transport machinery, we analyzed pin2 and pin2 abcb1 abcb19 mutants. Although these mutants are severely defective in shootward auxin transport and gravitropism, similar to aux1 (22), they did not show defects in lateral root formation, nor did they have a reduced sensitivity to IBA ( fig. S11, A to D), raising the possiblity that this reflux model requires the global features of the PIN and ABCB localization for directing auxin into the oscillation zone (23,24). We could find further evidence by using three chemically unrelated auxin transport inhibitors-1-N-naphtylphtalamic acid (NPA), 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid (BUM), and benzyloxy-IAA (BZ-IAA)-that target mainly ABCB-type transporters (NPA and BUM) (22,25) or generally interfere with AUX1-, PIN-, and ABCBbased auxin transport (BZ-IAA) (26). Consistent with our simulations, treatments with any of these inhibitors preserved the occurrence of PCD in the lateral root cap (fig. S11E) but resulted in ectopic DR5 activity in the lateral root cap and epidermis (Fig. 4, C and D; fig. S11, F and G; and movie S8), as well as impaired DR5:Luciferase oscillations (movie S9) and lateral root formation ( Fig. 4E and fig. S11, H and I), corroborating the auxin reflux model (27). Although we could not completely resolve the molecular mechanism for auxin efflux at present, our data underscore the necessity of auxin transport in the coordination of PCD in the most distal lateral root cap cells with oscillatory gene expression in the oscillation zone for lateral root spacing ( fig. S12).
During the exploration of the soil, root tips sense, through the root cap, the nutrient and water status of the soil they are traversing, as well as obstacles they may encounter (6,28). Transduction of that information may serve to control the periodicity of programmed cell death, thus altering the frequency of lateral root development. In this way, root systems may adjust development according to the quality of the soils they are passing through.