New insights in shoot apical meristem morphogenesis: Isotropy comes into play

Abstract

The great complexity and plasticity of aerial plant shapes largely results from the activity of the shoot apical meristem (SAM), a group of undifferentiated cells which produces all the aboveground organs of the plant. Organogenesis at the SAM is regulated by the hormone auxin, which, through an integration of active transport, signalling and transcriptional regulation, determines the positional and temporal information dictating where, when, and how a new organ will be formed. At the cellular level, the information stemming from the regulatory molecular networks influences the growth of the cells within the tissue to give rise to the final organ shape. The growth of plant cells is mainly controlled by the cell wall, a rigid structure mainly made of polysaccharides, which surrounds the cells and links them together in an organismal continuum. Over the years, several lines of evidence have pointed at a role for the regulation of the elasticity of the cell wall, downstream of auxin action, in the formation of organs at the SAM. We have recently shown that auxin also induces a shift toward isotropic growth by modulating the organization of cortical microtubules in peripheral SAM cells, which promotes organ formation. Here, we discuss our results and identify new hypotheses to drive future research.

The formation of lateral organs at the SAM depends on the plant hormone auxin.¹ Auxin accumulation in discrete cell foci at the SAM periphery, through an active transport mechanism mediated by the PIN-FORMED 1 (PIN1) efflux carrier, induces the initiation of new organs.^2-5 The accumulation of auxin at the SAM periphery is known to activate AUXIN RESPONSE FACTOR (ARF)-mediated transcriptional responses which are required for organ formation.^6,7 Beyond these transcriptional responses, it is still not clear how auxin modifies cell growth to promote organ bulging. The growth of plant cells is known to be regulated by the cell wall that counteracts the vacuole-borne turgor pressure. Plant cell growth occurs when the cell wall irreversibly yields to the inner turgor pressure. In the context of an expanding tissue, the mechanical properties of the cell wall must be fine-tuned to promote and direct the collective growth of the cells but at the same time to prevent the breakage of the tissue.⁸ Auxin has been known since long time to promote cell growth by enhancing the extensibility of the cell wall through acidification and further activation of wall-loosening proteins.⁹ Thus it is not surprising that in the last few years several research efforts have been made to address the role of the mechanical properties of the cell wall in the context of SAM organogenesis. The development of new micro-indentation techniques to measure cell wall mechanical properties (atomic force microscopy; AFM) and of new quantitative imaging methods to measure growth associated with numerical simulations have independently led to the finding that differences in cell wall rigidity at the SAM do exist and are associated to differential growth.^10-12 Indeed, cells at the periphery of the SAM, where new organs are formed, turned out to be more elastic (i.e. with less rigid cell wall) and faster-growing than cells at the meristem apex that does not generate organs.^10,11 Relevantly, higher cell wall elasticity and cell growth rates of the SAM peripheral zone overlap with the expression domains of several ARF transcription factors,⁷ further pointing at a role for auxin-mediated transcriptional responses in determining the mechanical readout of the tissue. A compelling amount of evidence shows that auxin regulates the expression of several genes encoding cell wall-loosening proteins such as expansins and pectin methylesterases (PMEs) among the others, as well as of SAUR genes whose function is required for cell wall acidification.^13-18 Relevantly, localized, exogenous applications or transgenic expression of expansins or PMEs have been shown to promote the formation of organs or organ-like structures, further confirming that changes of cell wall mechanical properties underlie SAM morphogenesis.^12,19-22

We recently demonstrated that the increase in cell wall elasticity is not the only process accounting for the changes in growth direction that occur when the newly-formed organs grow away from the meristem surface. Indeed we provided evidence that the initiation of a new growth axis at the meristem side is caused by an auxin-induced change in directional cell expansion – i.e. anisotropy.²³ At the cellular level, growth anisotropy is determined by the ordered alignment of cellulose microfibrils around the cell, allowing cell expansion preferentially along the axis perpendicular to the main microfibril orientation. Conversely, the random arrangement of cellulose microfibrils leads to cell expansion in all directions, i.e. isotropy. At the tissue levels, the degree of anisotropy is globally determined by the uniformity of microfibril alignment between adjacent cells across the tissue.²⁴ The orientation of cellulose microfibrils strictly depends on the cortical microtubule (CMT) network, which guides the insertion of cellulose synthase complexes and directs their further movement in the plasma membrane, to guide cellulose deposition in the cell wall.^25-27 Previous works using CMT orientation as a bona fide indicator of cell and tissue growth anisotropy, demonstrated that the SAM displays intrinsic growth anisotropy.^28,29 CMT orientation in the peripheral SAM cells displays a specific supracellular circumferential alignment that is better highlighted when organ formation is inhibited by blocking polar auxin transport.²⁸ In the absence of auxin accumulation, circumferential CMT alignment at the SAM directs global cell growth along the vertical growth axis of the stem, by restricting lateral growth.^28,30 We have shown that auxin removes this circumferential growth constraint by disrupting the anisotropic organization of CMTs in peripheral SAM cells at the early stages of organogenesis, promoting the emergence of lateral organs later on.²³ The disruption of CMT anisotropy is per se sufficient to promote organ outgrowth, as demonstrated by experiments in which chemical or genetic alterations of CMT organization were able to promote lateral outgrowth formation even in absence of auxin transport.²³ How does this breaking of SAM anisotropy functionally relate to cell wall loosening? Numerical simulations, using a virtual 3D model of the meristem, provided plausible theoretical evidence that the shift toward isotropic growth functionally interacts with cell wall loosening to promote organ formation. Indeed the model was only able to reproduce the initiation of realistic organ primordia when physiological levels of cell wall loosening (determined by means of AFM on auxin-treated meristem) were associated with a shift towards isotropic growth.²³ By contrast, when the model was instructed to loosen cell walls locally while maintaining growth anisotropy, the virtual meristem gave rise to a stem that bent away from the loosened spot instead of forming an outgrowth.²³ In vivo, loosening-mediated bending was shown to be associated with phototropic responses where a localized, auxin-induced expression of expansins precede changes in hypocotyl direction.¹⁵ This suggests that plants may discriminate the generation of a new growth axis from a change in growth direction of the existing one by imposing isotropic growth onto localized cell wall loosening.

The synergistic action of cell wall loosening and growth isotropy in SAM cells must be tightly regulated to promote organ formation efficiently. Auxin might play a central role in synchronizing the concurrent changes in cell growth rates and direction through the bifurcation of its signalling pathway. As discussed above, the expression of a number of genes promoting cell wall loosening is under the control of ARF-mediated transcriptional responses activated by the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALLING F-BOX (TIR1/ABF) auxin receptors.^13-18 By contrast, we found that the auxin-mediated regulation of CMT organization at the SAM is under the control of the AUXIN BINDING PROTEIN 1 (ABP1) extracellular receptor.²³ ABP1 was previously demonstrated to regulate CMT organization through a pathway involving RHO GTPASE of PLANT 6 (ROP6) and its interacting protein ROP INTERACTING CRIB MOTIF-CONTAINING PROTEIN1 (RIC1), which in turn affects the CMT-ordering activity of KATANIN 1 (KTN1).^23,31-33 The bifurcation of auxin signalling into two separate and spatially distinct pathways could be a way to provide robustness to SAM organogenesis. However, the role of ABP1 in auxin-mediated development has been recently put into discussion by the identification of novel knock-out alleles that do not display embryo lethality³⁴ a trait classically associated with the loss of ABP1 function.³⁵ Although no conclusive proof ruling out ABP1 function in the regulation of CMT organization has been provided so far, the role of this protein in the SAM must be carefully assessed in the future. It is interesting to point out that the auxin-mediated disorganization of CMT at the SAM seems to occur at least in part independently from auxin-regulated transcriptional responses as we could not observe precise spatial overlap between the two responses both in vivo,²³ or in auxin-treated meristem grown in vitro (Fig. 1).

Figure 1.

Lack of spatial overlap between auxin-mediated CMT disorganization and transcriptional responses in SAM cells. An NPA-grown 35S:GFP-MBD DR5: VENUS meristem was imaged before (t = 0) and 24 h after the beginning of the auxin treatment. The green channel displays CMT orientation as inferred by the 35S:GFP-MBD marker. The red channel display DR5: VENUS expression (nuclear signal) and chlorophyll autofluorescence background. Note that in the auxin-treated meristem DR5:VENUS expression is limited to few cells whereas the disorganization of CMT encompasses the entire SAM surface. The experimental and imaging setup were previously described.²³

The function of anisotropy-to-isotropy shifts at the SAM, might not be solely related to organ outgrowth but might play a role also in organ patterning by interacting with the auxin transport system. Previous evidence showed that the polarity of PIN1 at the SAM surface negatively correlates with anisotropic CMT organization, with PIN1 apparently being excluded by the plasma membranes associated with CMTs.³⁶ Disruption of CMT organization has been shown to perturb the coordination of PIN1 polarity at the tissue level,³⁷ likely due to the presence of cellulosic connections between the cell wall and the membrane that limit lateral diffusion of the PIN efflux carriers.³⁸ In the SAM, the auxin-mediated disorganization of CMT occurs in organ-founder cells, where PIN1 localization on the plasma membrane becomes progressively apolar in order to promote the formation of auxin maxima at the site of organ initiation.^3-5 Relevantly, ROP6 GTPase has been previously associated with the regulation of both CMT organization^31,39 and PIN distribution,^40,41 further suggesting a coordinated control of the two pathways. Taken together, these lines of evidence would suggest the existence of a positive feedback between cell growth and auxin accumulation that could be of major relevance in the regulation of phyllotactic patterning of the SAM.

In conclusion, our work demonstrates the existence of a previously unrecognized mechanism based on the auxin-mediated regulation of growth anisotropy that underlies the formation of organs at the SAM of Arabidopsis thaliana. As discussed above, our results further suggest the existence of a complex web of regulatory cross-talks and feedbacks that might play a relevant role in fine tuning organ formation and meristem patterning, paving the way for future research.

Funding

The work of the authors was funded by grants from EraSysBio+ (iSAM) and ERC (Morpho-dynamics).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgements

We would like to thank all the colleagues that made our work on anisotropy a rewarding experience.