A cell wall-associated gene network shapes leaf boundary domains.

Boundary domains delimit and organize organ growth throughout plant development almost relentlessly, building plant architecture and morphogenesis. Boundary domains display reduced growth and orchestrate development of adjacent tissues in a non-cell-autonomous manner. How these two functions are achieved remains elusive despite the identification of several boundary-specific genes. Here, we show using morphometrics at the organ and cellular levels that leaf boundary domain development requires SPINDLY (SPY), an O-fucosyltransferase, to act as cell growth repressor. Furthermore, we show that SPY acts redundantly with the CUP-SHAPED COTYLEDON transcription factors (CUC2 and CUC3), which are major determinants of boundaries development. Accordingly, at the molecular level CUC2 and SPY repress a common set of genes involved in cell wall loosening, providing a molecular framework for the growth repression associated with boundary domains. Atomic force microscopy confirmed that young leaf boundary domain cells have stiffer cell walls than marginal outgrowth. This differential cell wall stiffness was reduced in spy mutant plants. Taken together, our data reveal a concealed CUC2 cell wall-associated gene network linking tissue patterning with cell growth and mechanics.

The preprophase band of microtubules controls the robustness of division orientation in plants.

Controlling cell division plane orientation is essential for morphogenesis in multicellular organisms. In plant cells, the future cortical division plane is marked before mitotic entry by the preprophase band (PPB). Here, we characterized an Arabidopsis trm (TON1 Recruiting Motif) mutant that impairs PPB formation but does not affect interphase microtubules. Unexpectedly, PPB disruption neither abolished the capacity of root cells to define a cortical division zone nor induced aberrant cell division patterns but rather caused a loss of precision in cell division orientation. Our results advocate for a reassessment of PPB function and division plane determination in plants and show that a main output of this microtubule array is to limit spindle rotations in order to increase the robustness of cell division.

A protein phosphatase 2A complex spatially controls plant cell division.

In the absence of cell migration, the orientation of cell divisions is crucial for body plan determination in plants. The position of the division plane in plant cells is set up premitotically via a transient cytoskeletal array, the preprophase band, which precisely delineates the cortical plane of division. Here we describe a protein complex that targets protein phosphatase 2A activity to microtubules, regulating the transition from the interphase to the premitotic microtubule array. This complex, which comprises TONNEAU1 and a PP2A heterotrimeric holoenzyme with FASS as regulatory subunit, is recruited to the cytoskeleton via the TONNEAU1-recruiting motif family of proteins. Despite the acentrosomal nature of plant cells, all members of this complex share similarity with animal centrosomal proteins involved in ciliary and centriolar/centrosomal functions, revealing an evolutionary link between the cortical cytoskeleton of plant cells and microtubule organizers in other eukaryotes.

The Arabidopsis TRM1-TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes.

Land plant cells assemble microtubule arrays without a conspicuous microtubule organizing center like a centrosome. In Arabidopsis thaliana, the TONNEAU1 (TON1) proteins, which share similarity with FOP, a human centrosomal protein, are essential for microtubule organization at the cortex. We have identified a novel superfamily of 34 proteins conserved in land plants, the TON1 Recruiting Motif (TRM) proteins, which share six short conserved motifs, including a TON1-interacting motif present in all TRMs. An archetypal member of this family, TRM1, is a microtubule-associated protein that localizes to cortical microtubules and binds microtubules in vitro. Not all TRM proteins can bind microtubules, suggesting a diversity of functions for this family. In addition, we show that TRM1 interacts in vivo with TON1 and is able to target TON1 to cortical microtubules via its C-terminal TON1 interaction motif. Interestingly, three motifs of TRMs are found in CAP350, a human centrosomal protein interacting with FOP, and the C-terminal M2 motif of CAP350 is responsible for FOP recruitment at the centrosome. Moreover, we found that TON1 can interact with the human CAP350 M2 motif in yeast. Taken together, our results suggest conservation of eukaryotic centrosomal components in plant cells.

The function of TONNEAU1 in moss reveals ancient mechanisms of division plane specification and cell elongation in land plants.

The preprophase band (PPB) is a transient ring of microtubules that forms before mitosis in land plants, and delineates the cytokinetic division plane established at telophase. It is one of the few derived traits specific to embryophytes, in which it is involved in the spatial control of cell division. Here we show that loss of function of Physcomitrella patens PpTON1 strongly affects development of the moss gametophore, phenocopying the developmental syndrome observed in Arabidopsis ton1 mutants: mutant leafy shoots display random orientation of cell division and severe defects in cell elongation, which are correlated with absence of PPB formation and disorganization of the cortical microtubule array in interphase cells. In hypomorphic Ppton1 alleles, PPB are still formed, whereas elongation defects are observed, showing the dual function of TON1 in organizing cortical arrays of microtubules during both interphase and premitosis. Ppton1 mutation has no impact on development of the protonema, which is consistent with the documented absence of PPB formation at this stage, apart from alteration of the gravitropic response, uncovering a new function of TON1 proteins in plants. Successful reciprocal cross-complementation between Physcomitrella and Arabidopsis shows conservation of TON1 function during land plant evolution. These results establish the essential role of the PPB in division plane specification in a basal land plant lineage, and provide new information on the function of TON1. They point to an ancient mechanism of cytoskeletal control of division plane positioning and cell elongation in land plants.

Branching genes are conserved across species. Genes controlling a novel signal in pea are coregulated by other long-distance signals.

Physiological and genetic studies with the ramosus (rms) mutants in garden pea (Pisum sativum) and more axillary shoots (max) mutants in Arabidopsis (Arabidopsis thaliana) have shown that shoot branching is regulated by a network of long-distance signals. Orthologous genes RMS1 and MAX4 control the synthesis of a novel graft-transmissible branching signal that may be a carotenoid derivative and acts as a branching inhibitor. In this study, we demonstrate further conservation of the branching control system by showing that MAX2 and MAX3 are orthologous to RMS4 and RMS5, respectively. This is consistent with the long-standing hypothesis that branching in pea is regulated by a novel long-distance signal produced by RMS1 and RMS5 and that RMS4 is implicated in the response to this signal. We examine RMS5 expression and show that it is more highly expressed relative to RMS1, but under similar transcriptional regulation as RMS1. Further expression studies support the hypothesis that RMS4 functions in shoot and rootstock and participates in the feedback regulation of RMS1 and RMS5 expression. This feedback involves a second novel long-distance signal that is lacking in rms2 mutants. RMS1 and RMS5 are also independently regulated by indole-3-acetic acid. RMS1, rather than RMS5, appears to be a key regulator of the branching inhibitor. This study presents new interactions between RMS genes and provides further evidence toward the ongoing elucidation of a model of axillary bud outgrowth in pea.

Gamma-tubulin is essential for microtubule organization and development in Arabidopsis.

The process of microtubule nucleation in plant cells is still a major question in plant cell biology. gamma-Tubulin is known as one of the key molecular players for microtubule nucleation in animal and fungal cells. Here, we provide genetic evidence that in Arabidopsis thaliana, gamma-tubulin is required for the formation of spindle, phragmoplast, and cortical microtubule arrays. We used a reverse genetics approach to investigate the role of the two Arabidopsis gamma-tubulin genes in plant development and in the formation of microtubule arrays. Isolation of mutants in each gene and analysis of two combinations of gamma-tubulin double mutants showed that the two genes have redundant functions. The first combination is lethal at the gametophytic stage. Disruption of both gamma-tubulin genes causes aberrant spindle and phragmoplast structures and alters nuclear division in gametophytes. The second combination of gamma-tubulin alleles affects late seedling development, ultimately leading to lethality 3 weeks after germination. This partially viable mutant combination enabled us to follow dynamically the effects of gamma-tubulin depletion on microtubule arrays in dividing cells using a green fluorescent protein marker. These results establish the central role of gamma-tubulin in the formation and organization of microtubule arrays in Arabidopsis.

The branching gene RAMOSUS1 mediates interactions among two novel signals and auxin in pea.

In Pisum sativum, the RAMOSUS genes RMS1, RMS2, and RMS5 regulate shoot branching via physiologically defined mobile signals. RMS1 is most likely a carotenoid cleavage enzyme and acts with RMS5 to control levels of an as yet unidentified mobile branching inhibitor required for auxin inhibition of branching. Our work provides molecular, genetic, and physiological evidence that RMS1 plays a central role in a shoot-to-root-to-shoot feedback system that regulates shoot branching in pea. Indole-3-acetic acid (IAA) positively regulates RMS1 transcript level, a potentially important mechanism for regulation of shoot branching by IAA. In addition, RMS1 transcript levels are dramatically elevated in rms3, rms4, and rms5 plants, which do not contain elevated IAA levels. This degree of upregulation of RMS1 expression cannot be achieved in wild-type plants by exogenous IAA application. Grafting studies indicate that an IAA-independent mobile feedback signal contributes to the elevated RMS1 transcript levels in rms4 plants. Therefore, the long-distance signaling network controlling branching in pea involves IAA, the RMS1 inhibitor, and an IAA-independent feedback signal. Consistent with physiological studies that predict an interaction between RMS2 and RMS1, rms2 mutations appear to disrupt this IAA-independent regulation of RMS1 expression.

MAX4 and RMS1 are orthologous dioxygenase-like genes that regulate shoot branching in Arabidopsis and pea.

Shoot branching is inhibited by auxin transported down the stem from the shoot apex. Auxin does not accumulate in inhibited buds and so must act indirectly. We show that mutations in the MAX4 gene of Arabidopsis result in increased and auxin-resistant bud growth. Increased branching in max4 shoots is restored to wild type by grafting to wild-type rootstocks, suggesting that MAX4 is required to produce a mobile branch-inhibiting signal, acting downstream of auxin. A similar role has been proposed for the pea gene, RMS1. Accordingly, MAX4 and RMS1 were found to encode orthologous, auxin-inducible members of the polyene dioxygenase family.