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Plant cells continuously experience mechanical stress resulting from the cell wall that bears internal turgor pressure. Cortical microtubules align with the predicted maximal tensile stress direction to guide cellulose biosynthesis and therefore results in cell wall reinforcement. We have previously identified Increased Petal Growth Anisotropy (IPGA1) as a putative microtubule-associated protein in Arabidopsis, but the function of IPGA1 remains unclear. Here, using the Arabidopsis cotyledon pavement cell as a model, we demonstrated that IPGA1 forms protein granules and interacts with ANGUSTIFOLIA (AN) to cooperatively regulate microtubule organisation in response to stress. Application of mechanical perturbations, such as cell ablation, led to microtubule reorganisation into aligned arrays in wild-type cells. This microtubule response to stress was enhanced in the IPGA1 loss-of-function mutant. Mechanical perturbations promoted the formation of IPGA1 granules on microtubules. We further showed that IPGA1 physically interacted with AN both in vitro and on microtubules. The ipga1 mutant alleles exhibited reduced interdigitated growth of pavement cells, with smooth shape. IPGA1 and AN had a genetic interaction in regulating pavement cell shape. Furthermore, IPGA1 genetically and physically interacted with the microtubule-severing enzyme KATANIN. We propose that the IPGA1-AN module regulates microtubule organisation and pavement cell shape.

The organization of the microtubule cytoskeleton is critical for cell and organ morphogenesis. The evolutionarily conserved microtubule-severing enzyme KATANIN plays critical roles in microtubule organization in the plant and animal kingdoms. We previously used conical cell of Arabidopsis thaliana petals as a model system to investigate cortical microtubule organization and cell morphogenesis and determined that KATANIN promotes the formation of circumferential cortical microtubule arrays in conical cells. Here, we demonstrate that the conserved protein phosphatase PP2A interacts with and dephosphorylates KATANIN to promote the formation of circumferential cortical microtubule arrays in conical cells. KATANIN undergoes cycles of phosphorylation and dephosphorylation. Using co-immunoprecipitation coupled with mass spectrometry, we identified PP2A subunits as KATANIN-interacting proteins. Further biochemical studies showed that PP2A interacts with and dephosphorylates KATANIN to stabilize its cellular abundance. Similar to the katanin mutant, mutants for genes encoding PP2A subunits showed disordered cortical microtubule arrays and defective conical cell shape. Taken together, these findings identify PP2A as a regulator of conical cell shape and suggest that PP2A mediates KATANIN phospho-regulation during plant cell morphogenesis.

Sclerotinia sclerotiorum is a devastating pathogen that infects a broad range of host plants. The mechanism underlying plant defence against fungal invasion is still not well characterized. Here, we report that ANGUSTIFOLIA (AN), a CtBP family member, plays a role in the defence against S. sclerotiorum attack. Arabidopsis an mutants exhibited stronger resistance to S. sclerotiorum at the early stage of infection than wild-type plants. Accordingly, an mutants exhibited stronger activation of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses, including mitogen-activated protein kinase activation, reactive oxygen species accumulation, callose deposition, and the expression of PTI-responsive genes, upon treatment with PAMPs/microbe-associated molecular patterns. Moreover, Arabidopsis lines overexpressing AN were more susceptible to S. sclerotiorum and showed defective PTI responses. Our luminometry, bimolecular fluorescence complementation, coimmunoprecipitation, and in vitro pull-down assays indicate that AN interacts with allene oxide cyclases (AOC), essential enzymes involved in jasmonic acid (JA) biosynthesis, negatively regulating JA biosynthesis in response to S. sclerotiorum infection. This work reveals AN is a negative regulator of the AOC-mediated JA signalling pathway and PTI activation.

Root hydrotropism refers to root directional growth toward soil moisture. Cortical microtubule arrays are essential for determining the growth axis of the elongating cells in plants. However, the role of microtubule reorganization in root hydrotropism remains elusive. Here, we demonstrate that the well-ordered microtubule arrays and the microtubule-severing protein KATANIN (KTN) play important roles in regulating root hydrotropism in Arabidopsis. We found that the root hydrotropic bending of the ktn1 mutant was severely attenuated but not root gravitropism. After hydrostimulation, cortical microtubule arrays in cells of the elongation zone of wild-type (WT) Col-0 roots were reoriented from transverse into an oblique array along the axis of cell elongation, whereas the microtubule arrays in the ktn1 mutant remained in disorder. Moreover, we revealed that abscisic acid (ABA) signaling enhanced the root hydrotropism of WT and partially rescued the oryzalin (a microtubule destabilizer) alterative root hydrotropism of WT but not ktn1 mutants. These results suggest that katanin-dependent microtubule ordering is required for root hydrotropism, which might work downstream of ABA signaling pathways for plant roots to search for water.

Unlike animal cells, plant cells do not possess centrosomes that serve as microtubule organizing centers; how microtubule arrays are organized throughout plant morphogenesis remains poorly understood. We report here that Arabidopsis INCREASED PETAL GROWTH ANISOTROPY 1 (IPGA1), a previously uncharacterized microtubule-associated protein, regulates petal growth and shape by affecting cortical microtubule organization. Through a genetic screen, we showed that IPGA1 loss-of-function mutants displayed a phenotype of longer and narrower petals, as well as increased anisotropic cell expansion of the petal epidermis in the late phases of flower development. Map-based cloning studies revealed that IPGA1 encodes a previously uncharacterized protein that colocalizes with and directly binds to microtubules. IPGA1 plays a negative role in the organization of cortical microtubules into parallel arrays oriented perpendicular to the axis of cell elongation, with the ipga1-1 mutant displaying increased microtubule ordering in petal abaxial epidermal cells. The IPGA1 family is conserved among land plants and its homologs may have evolved to regulate microtubule organization. Taken together, our findings identify IPGA1 as a novel microtubule-associated protein and provide significant insights into IPGA1-mediated microtubule organization and petal growth anisotropy., Arabidopsis IPGA1 is a previously uncharacterized microtubule-associated protein that functions in modulating cell and petal shape through its effect on cortical microtubule organization in abaxial petal epidermal cells.

Cryptochromes (CRYs) are photoreceptors or components of the molecular clock in various evolutionary lineages, and they are commonly regulated by polyubiquitination and proteolysis. Multiple E3 ubiquitin ligases regulate CRYs in animal models, and previous genetics study also suggest existence of multiple E3 ubiquitin ligases for plant CRYs. However, only one E3 ligase, Cul4COP1/SPAs, has been reported for plant CRYs so far. Here we show that Cul3LRBs is the second E3 ligase of CRY2 in Arabidopsis. We demonstrate the blue light-specific and CRY-dependent activity of LRBs (Light-Response Bric-a-Brack/Tramtrack/Broad 1, 2 & 3) in blue-light regulation of hypocotyl elongation. LRBs physically interact with photoexcited and phosphorylated CRY2, at the CCE domain of CRY2, to facilitate polyubiquitination and degradation of CRY2 in response to blue light. We propose that Cul4COP1/SPAs and Cul3LRBs E3 ligases interact with CRY2 via different structure elements to regulate the abundance of CRY2 photoreceptor under different light conditions, facilitating optimal photoresponses of plants grown in nature., The fate of proteins in cells is determined by not only synthesis but also degradation. Here Chen et al. show that degradation of the plant blue light receptor CRY2 is determined by two distinct E3 ubiquitin ligases, Cul4COP1/SPAs and Cul3LRBs, regulating the function of CRY2 under different light conditions.

Cryptochromes (CRYs) are photoreceptors or components of the molecular clock in various evolutionary lineages, and they are commonly regulated by polyubiquitination and proteolysis. Multiple E3 ubiquitin ligases regulate CRYs in animal models, and previous genetics study also suggest existence of multiple E3 ubiquitin ligases for plant CRYs. However, only one E3 ligase, Cul4COP1-SPAs, has been reported for plant CRYs so far. Here we show that Cul3LRBs is the second E3 ligase of CRY2 in Arabidopsis. We demonstrated the blue light-specific and CRY-dependent activity of LRBs (Light-Response Bric-a-Brack/Tramtrack/Broad 1, 2 & 3) in blue-light regulation of hypocotyl elongation. LRBs physically interact with photoexcited and phosphorylated CRY2 to facilitate polyubiquitination and degradation of CRY2 in response to blue light. We propose that Cul4COP1-SPAs and Cul3LRBs E3 ligases interact with CRY2 via different structure elements to regulate the abundance of CRY2 photoreceptor under different light conditions, facilitating optimal photoresponses of plants grown in nature.

The microtubule cytoskeleton plays an important role in cell shape and plant development. During the past decades, the ability to use confocal microcopy to observe microtubules in living cells using fluorescent protein fusions has given plant scientists the opportunity to answer outstanding biological questions. Plants contain diverse epidermal cells with distinct morphologies and physiological functions. For example, flowering plants have specialized petal conical cells that likely facilitate functions such as providing grips for bee pollinators. Here, we summarize recent progress on live imaging of the microtubule cytoskeleton in conical cells. Firstly, we present a simple method for live-cell confocal imaging of conical cells, which is suitable for the quantification of the cell geometry. Secondly, we describe a method for observing microtubule organization in conical cells of Arabidopsis thaliana expressing green fluorescent protein (GFP)-tagged α-tubulin 6 (GFP-TUA6). These live imaging approaches are likely to lead to rapid advances in our knowledge of the role of microtubules in conical cell shaping.

Proper flower development is essential for sexual reproductive success and the setting of fruits and seeds. The availability of a high quality genome sequence for pineapple makes it an excellent model for studying fruit and floral organ development. In this study, we sequenced 27 different pineapple floral samples and integrated nine published RNA-seq datasets to generate tissue- and stage-specific transcriptomic profiles. Pairwise comparisons and weighted gene co-expression network analysis successfully identified ovule-, stamen-, petal- and fruit-specific modules as well as hub genes involved in ovule, fruit and petal development. In situ hybridization confirmed the enriched expression of six genes in developing ovules and stamens. Mutant characterization and complementation analysis revealed the important role of the subtilase gene AcSBT1.8 in petal development. This work provides an important genomic resource for functional analysis of pineapple floral organ growth and fruit development and sheds light on molecular networks underlying pineapple reproductive organ growth., Wang et al. perform RNA-Seq on pineapple floral samples and also use previously published RNA-Seq datasets to generate tissue- and stage-specific transcriptomic profiles. The authors use weighted gene co-expression network analysis to identify gene networks, bringing insight to underlying pineapple reproductive organ growth.

The microtubule cytoskeleton plays an important role in cell shape and plant development. During the past decades, the ability to use confocal microcopy to observe microtubules in living cells using fluorescent protein fusions has given plant scientists the opportunity to answer outstanding biological questions. Plants contain diverse epidermal cells with distinct morphologies and physiological functions. For example, flowering plants have specialized petal conical cells that likely facilitate functions such as providing grips for bee pollinators. Here, we summarize recent progress on live imaging of the microtubule cytoskeleton in conical cells. Firstly, we present a simple method for live-cell confocal imaging of conical cells, which is suitable for the quantification of the cell geometry. Secondly, we describe a method for observing microtubule organization in conical cells of Arabidopsis thaliana expressing green fluorescent protein (GFP)-tagged α-tubulin 6 (GFP-TUA6). These live imaging approaches are likely to lead to rapid advances in our knowledge of the role of microtubules in conical cell shaping.

Compressive sensing theory mainly includes the sparsely of signal processing, the structure of the measurement matrix and reconstruction algorithm. Reconstruction algorithm is the core content of CS theory, that is, through the low dimensional sparse signal recovers the original signal accurately. This thesis based on the theory of CS to study further on seismic data reconstruction algorithm. We select Orthogonal Matching Pursuit algorithm as a base reconstruction algorithm. Then do the specific research for the implementation principle, the structure of the algorithm of AOMP and make the signal simulation at the same time. In view of the OMP algorithm reconstruction speed is slow and the problems need to be a given number of iterations, which developed an improved scheme. We combine the optimized OMP algorithm of constraint the optimal matching of item selection strategy, the backwards gradient projection ideas of adaptive variance step gradient projection method and the original algorithm to improve it. Simulation experiments show that improved OMP algorithm is superior to traditional OMP algorithm of improvement in the reconstruction time and effect under the same condition.

Megasporogenesis is a key step during ovule development in angiosperms, but the small number and inaccessibility of these cells have hampered molecular and genome-wide studies. Thus, many questions remain regarding the molecular basis of cell specification, differentiation, and development in the female gametophyte. Here, taking advantage of the correlation between spikelet length and ovule development in rice (Oryza sativa), we studied the transcriptome dynamics of young ovules at three stages, the archesporial cell, the megaspore mother cell before meiosis, and the functional megaspore after meiosis, using expression profiling based on RNA sequencing. Our analysis showed that 5,274 genes were preferentially expressed in ovules during megasporogenesis as compared to ovules at the mature female gametophyte stage. Out of these, 958 (18.16%) genes were archesporial cell- and/or megaspore mother cell-preferential genes, and represent a significant enrichment of genes involved in hormone signal transduction and plant pathogen interaction pathways, as well as genes encoding transcription factors. The expression patterns of nine genes that were preferentially expressed in ovules of different developmental stages, including the OsERECTA2 (OsER2) receptor-like kinase gene, were confirmed by in situ hybridization. We further characterized the OsER2 loss-of-function mutant, which had an excessive number of female germline cells and an abnormal female gametophyte, suggesting that OsER2 regulates germline cell specification during megasporogenesis in rice. These results expand our understanding of the molecular control of megasporogenesis in rice and contribute to the functional studies of genes involved in megasporogenesis.

Cortical microtubules guide the direction and deposition of cellulose microfibrils to build the cell wall, which in turn influences cell expansion and plant morphogenesis. In the model plant Arabidopsis thaliana (Arabidopsis), petal is a relatively simple organ that contains distinct epidermal cells, such as specialized conical cells in the adaxial epidermis and relatively flat cells with several lobes in the abaxial epidermis. In the past two decades, the Arabidopsis petal has become a model experimental system for studying cell expansion and organ morphogenesis, because petals are dispensable for plant growth and reproduction. Recent advances have expanded the role of microtubule organization in modulating petal anisotropic shape formation and conical cell shaping during petal morphogenesis. Here, we summarize recent studies showing that in Arabidopsis, several genes, such as SPIKE1, Rho of plant (ROP) GTPases, and IPGA1, play critical roles in microtubule organization and cell expansion in the abaxial epidermis during petal morphogenesis. Moreover, we summarize the live-confocal imaging studies of Arabidopsis conical cells in the adaxial epidermis, which have emerged as a new cellular model. We discuss the microtubule organization pattern during conical cell shaping. Finally, we propose future directions regarding the study of petal morphogenesis and conical cell shaping.

Cortical microtubules guide the direction and deposition of cellulose microfibrils to build the cell wall, which in turn influences cell expansion and plant morphogenesis. In the model plant Arabidopsis thaliana (Arabidopsis), petal is a relatively simple organ that contains distinct epidermal cells, such as specialized conical cells in the adaxial epidermis and relatively flat cells with several lobes in the abaxial epidermis. In the past two decades, the Arabidopsis petal has become a model experimental system for studying cell expansion and organ morphogenesis, because petals are dispensable for plant growth and reproduction. Recent advances have expanded the role of microtubule organization in modulating petal anisotropic shape formation and conical cell shaping during petal morphogenesis. Here, we summarize recent studies showing that in Arabidopsis, several genes, such as SPIKE1, Rho of plant (ROP) GTPases, and IPGA1, play critical roles in microtubule organization and cell expansion in the abaxial epidermis during petal morphogenesis. Moreover, we summarize the live-confocal imaging studies of Arabidopsis conical cells in the adaxial epidermis, which have emerged as a new cellular model. We discuss the microtubule organization pattern during conical cell shaping. Finally, we propose future directions regarding the study of petal morphogenesis and conical cell shaping.

International audience; Root hairs are protrusions from root epidermal cells with crucial roles in plant soil interactions. Although much is known about patterning, polarity and tip growth of root hairs, contributions of membrane trafficking to hair initiation remain poorly understood. Here, we demonstrate that the trans-Golgi network-localized YPT-INTERACTING PROTEIN 4a and YPT-INTERACTING PROTEIN 4b (YIP4a/b) contribute to activation and plasma membrane accumulation of Rho-of-plant (ROP) small GTPases during hair initiation, identifying YIP4a/b as central trafficking components in ROP-dependent root hair formation.

Summary: The flowers of angiosperm species typically contain specialized conical cells. Although substantial progress has been achieved regarding the mechanisms underlying flower development, little is known about how petal cells achieve final conical shape. Here, we use 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) as a fluorescent pH indicator for analyzing the apoplastic pH of conical cells in Arabidopsis and show that normal conical cell expansion requires auxin signaling and apoplastic pH changes. By combining imaging analysis and genetic and pharmacological experiments, we demonstrate that apoplastic acidification and alkalization correlate with an increase and decrease in tip sharpening of conical cells, respectively. Initial expansion of conical cells is accompanied by decreased apoplastic pH, which is associated with increased auxin signaling. Decreased auxin levels, transport, or signaling abolishes cell wall acidification and causes reduced tip sharpening and heights of conical cells. These findings provide an insight into apoplastic pH regulation of conical cell expansion. : Arabidopsis adaxial epidermis contains specialized conical cells. Utilizing HPTS as a fluorescent pH indicator, Dang et al. demonstrate that conical cell expansion correlates with pH changes. They also demonstrate that auxin activity correlates with pH alternation and a variation in height of the cell and angle of its tip. Keywords: auxin, apoplastic pH, conical cell, cell wall acidification, anisotropic cell expansion

Plants have evolved diverse cell types with distinct sizes, shapes, and functions. For example, most flowering plants contain specialized petal conical epidermal cells that are thought to attract pollinators and influence light capture and reflectance, but the molecular mechanisms controlling conical cell shaping remain unclear. Here, through a genetic screen in Arabidopsis thaliana, we demonstrated that loss-of-function mutations in ANGUSTIFOLIA (AN), which encodes for a homolog of mammalian CtBP/BARs, displayed conical cells phenotype with wider tip angles, correlating with increased accumulation of reactive oxygen species (ROS). We further showed that exogenously supplied ROS generated similar conical cell phenotypes as the an mutants. Moreover, reduced endogenous ROS levels resulted in deceased tip sharpening of conical cells. Furthermore, through enhancer screening, we demonstrated that mutations in katanin (KTN1) enhanced conical cell phenotypes of the an-t1 mutants. Genetic analyses showed that AN acted in parallel with KTN1 to control conical cell shaping. Both increased or decreased ROS levels and mutations in AN suppressed microtubule organization into well-ordered circumferential arrays. We demonstrated that the AN-ROS pathway jointly functioned with KTN1 to modulate microtubule ordering, correlating with the tip sharpening of conical cells. Collectively, our findings revealed a mechanistic insight into ROS homeostasis regulation of microtubule organization and conical cell shaping., Author summary Plants have diverse cell types with distinct sizes, shapes, and functions. For example, most flowering plants contain specialized conical petal epidermal cells that are thought to attract bee pollinators, but the molecular mechanisms controlling conical cell shaping remain unclear. Here, through a genetic screen in Arabidopsis thaliana, we demonstrated that loss-of-function mutations in ANGUSTIFOLIA (AN), which encodes a homolog of mammalian CtBP/BARs, resulted in swollen conical cells, correlating with increased accumulation of reactive oxygen species (ROS). Furthermore, through an enhancer screening, we demonstrated that mutations in katanin (KTN1) enhanced conical cell phenotypes of the an-t1 mutants. Genetic analyses showed that AN acted in parallel with KTN1 to control conical cell shaping. We demonstrated that the AN-ROS pathway jointly functioned with KTN1 to modulate microtubule organization, correlating with the tip sharpening of conical cells. Collectively, our findings revealed a mechanistic insight into ROS-mediated regulation of conical cell shaping.

Root hairs are protrusions from root epidermal cells with crucial roles in plant soil interactions. Although much is known about patterning, polarity and tip growth of root hairs, contributions of membrane trafficking to hair initiation remain poorly understood. Here, we demonstrate that the trans-Golgi network-localized YPT-INTERACTING PROTEIN 4a and YPT-INTERACTING PROTEIN 4b (YIP4a/b) contribute to activation and plasma membrane accumulation of Rho-of-plant (ROP) small GTPases during hair initiation, identifying YIP4a/b as central trafficking components in ROP-dependent root hair formation., Summary: YPT-interacting proteins 4a and 4b (YIP4a/b) contribute to activation and plasma membrane accumulation of Rho-of-plant (ROP) small GTPases during hair initiation, identifying YIP4a/b as central trafficking components in ROP-dependent root hair formation.

The physiological functions of epidermal cells are largely determined by their diverse morphologies. Most flowering plants have special conical-shaped petal epidermal cells that are thought to influence light capture and reflectance, and provide pollinator grips, but the molecular mechanisms controlling conical cell shape remain largely unknown. Here, we developed a live-confocal imaging approach to quantify geometric parameters of conical cells in Arabidopsis thaliana (A. thaliana). Through genetic screens, we identified katanin (KTN1) mutants showing a phenotype of decreased tip sharpening of conical cells. Furthermore, we demonstrated that SPIKE1 and Rho of Plants (ROP) GTPases were required for the final shape formation of conical cells, as KTN1 does. Live-cell imaging showed that wild-type cells exhibited random orientation of cortical microtubule arrays at early developmental stages but displayed a well-ordered circumferential orientation of microtubule arrays at later stages. By contrast, loss of KTN1 prevented random microtubule networks from shifting into well-ordered arrays. We further showed that the filamentous actin cap, which is a typical feature of several plant epidermal cell types including root hairs and leaf trichomes, was not observed in the growth apexes of conical cells during cell development. Moreover, our genetic and pharmacological data suggested that microtubules but not actin are required for conical cell shaping. Together, our results provide a novel imaging approach for studying petal conical cell morphogenesis and suggest that the spatio-temporal organization of microtubule arrays plays crucial roles in controlling conical cell shape., Author summary How cells achieve their final shapes is a fundamental question in biology. Most flowering plants have special conical-shaped petal epidermal cells that are thought to attract pollinators, but the molecular and genetic mechanisms that control conical cell shape remain unknown. In this study, we developed a live-confocal imaging approach for the quantitative study of conical cell morphogenesis. Through genetic screens, we showed that A. thaliana KTN1, ROP GTPases, and SPIKE1 are required for conical cell shaping. Live-cell imaging showed that loss of KTN1 prevented random microtubule networks from shifting into well-ordered microtubule arrays at later developmental stages, which is correlated with the tip sharpening of conical cells. Moreover, genetic and pharmacological data suggested that microtubules but not actin are required for conical cell shaping. Together, our findings provide significant insights into the spatio-temporal organization of microtubules that controls conical cell development.

In multicellular plant organs, cell shape formation depends on molecular switches to transduce developmental or environmental signals and to coordinate cell-to-cell communication. Plants have a specific subfamily of the Rho GTPase family, usually called Rho of Plants (ROP), which serve as a critical signal transducer involved in many cellular processes. In the last decade, important advances in the ROP-mediated regulation of plant cell morphogenesis have been made by using Arabidopsis thaliana leaf and cotyledon pavement cells. Especially, the auxin-ROP signaling networks have been demonstrated to control interdigitated growth of pavement cells to form jigsaw-puzzle shapes. Here, we review findings related to the discovery of this novel auxin-signaling mechanism at the cell surface. This signaling pathway is to a large extent independent of the well-known Transport Inhibitor Response (TIR)-Auxin Signaling F-Box (AFB) pathway, and instead requires Auxin Binding Protein 1 (ABP1) interaction with the plasma membrane-localized, transmembrane kinase (TMK) receptor-like kinase to regulate ROP proteins. Once activated, ROP influences cytoskeletal organization and inhibits endocytosis of the auxin transporter PIN1. The present review focuses on ROP signaling and its self-organizing feature allowing ROP proteins to serve as a bustling signal decoder and integrator for plant cell morphogenesis.

Summary Background: Ordered cortical microtubule (MT) arrays play a critical role in the spatial control of cell division and expansion and are essential for plant growth, morphogenesis, and development. Various developmental, hormonal, andmechanical signals and a large number of MT-associated proteins are known to impact cortical MT organization, but the underlying mechanisms remain poorly understood. Our previous studies show that auxin signaling, which is mediated by the ROP6 Rho GTPase and its effector RIC1, promotes the ordering of cortical MTs in pavement cells, but it is unknown how RIC1 controls the organization of cortical MTs into well-ordered arrays. Results: Our genetic screens identified the conserved MTsevering protein katanin (KTN1) as a downstream component of the ROP6-RIC1 signaling pathway leading to well-ordered arrangement of cortical MTs. KTN1 and RIC1 proteins displayed overlapping localization. In vivo and in vitro studies showed that RIC1 physically interacts with and promotes the MT-severing activity of KTN1. Live-cell imaging reveals a role for RIC1 in promoting detachment of branched MTs that is known to rely on KTN1. Conclusion: We have demonstrated that a Rho GTPase signaling pathway regulates katanin-mediated MT severing in plant cells and uncovered an explicit regulatory mechanism underpinning the alignment and ordering of cortical MTs in plants. Our findings provide new insights into regulatory mechanisms underlying growth stimuli such as auxin promote the organization of cortical MTs into parallel arrays in plants.

Plant organ growth and final shape rely on cell proliferation and, particularly, on cell expansion that largely determines the visible growth of plant organs. Arabidopsis (Arabidopsis thaliana) petals serve as an excellent model for dissecting the coordinated regulation of patterns of cell expansion and organ growth, but the molecular signaling mechanisms underlying this regulation remain largely unknown. Here, we demonstrate that during the late petal development stages, SPIKE1 (SPK1), encoding a guanine nucleotide exchange factor, activates Rho of Plants (ROP) GTPase proteins (ROP2, ROP4, and ROP6) to affect anisotropic expansion of epidermal cells in both petal blades and claws, thereby affecting anisotropic growth of the petal and the final characteristic organ shape. The petals of SPK1 knockdown mutants were significantly longer but narrower than those of the wild type, associated with increased anisotropic expansion of epidermal cells at late development stages. In addition, ROP2, ROP4, and ROP6 are activated by SPK1 to promote the isotropic organization of cortical microtubule arrays and thus inhibit anisotropic growth in the petal. Both knockdown of SPK1 and multiple rop mutants caused highly ordered cortical microtubule arrays that were transversely oriented relative to the axis of cell elongation after development stage 11. Taken together, our results suggest a SPK1-ROP-dependent signaling module that influences anisotropic growth in the petal and defines the final organ shape.

In multicellular plant organs, cell shape formation depends on molecular switches to transduce developmental or environmental signals and to coordinate cell-to-cell communication. Plants have a specific subfamily of the Rho GTPase family, usually called Rho of Plants (ROP), which serve as a critical signal transducer involved in many cellular processes. In the last decade, important advances in the ROP-mediated regulation of plant cell morphogenesis have been made by using Arabidopsis thaliana leaf and cotyledon pavement cells. Especially, the auxin-ROP signaling networks have been demonstrated to control interdigitated growth of pavement cells to form jigsaw-puzzle shapes. Here, we review findings related to the discovery of this novel auxin-signaling mechanism at the cell surface. This signaling pathway is to a large extent independent of the well-known Transport Inhibitor Response (TIR)-Auxin Signaling F-Box (AFB) pathway, and instead requires Auxin Binding Protein 1 (ABP1) interaction with the plasma membrane-localized, transmembrane kinase (TMK) receptor-like kinase to regulate ROP proteins. Once activated, ROP influences cytoskeletal organization and inhibits endocytosis of the auxin transporter PIN1. The present review focuses on ROP signaling and its self-organizing feature allowing ROP proteins to serve as a bustling signal decoder and integrator for plant cell morphogenesis.

The puzzle piece-shaped Arabidopsis leaf pavement cells (PCs) with interdigitated lobes and indents is a good model system to investigate the mechanisms that coordinate cell polarity and shape formation within a tissue. Auxin has been shown to coordinate the interdigitation by activating ROP GTPase-dependent signaling pathways. To identify additional components or mechanisms, we screened for mutants with abnormal PC morphogenesis and found that cytokinin signaling regulates the PC interdigitation pattern. Reduction in cytokinin accumulation and defects in cytokinin signaling (such as in ARR7-over-expressing lines, the ahk3cre1 cytokinin receptor mutant, and the ahp12345 cytokinin signaling mutant) enhanced PC interdigitation, whereas over-production of cytokinin and over-activation of cytokinin signaling in an ARR20 over-expression line delayed or abolished PC interdigitation throughout the cotyledon. Genetic and biochemical analyses suggest that cytokinin signaling acts upstream of ROPs to suppress the formation of interdigitated pattern. Our results provide novel mechanistic understanding of the pathways controlling PC shape and uncover a new role for cytokinin signaling in cell morphogenesis.

SummaryThe dynamic spatial and temporal distribution of the crucial plant signaling molecule auxin is achieved by feedback coordination of auxin signaling and intercellular auxin transport pathways [1, 2]. Developmental roles of auxin have been attributed predominantly to its effect on transcription; however, an alternative pathway involving AUXIN BINDING PROTEIN1 (ABP1) has been proposed to regulate clathrin-mediated endocytosis in roots and Rho-like GTPase (ROP)-dependent pavement cell interdigitation in leaves [3, 4]. In this study, we show that ROP6 and its downstream effector RIC1 regulate clathrin association with the plasma membrane for clathrin-mediated endocytosis, as well as for its feedback regulation by auxin. Genetic analysis revealed that ROP6/RIC1 acts downstream of ABP1 to regulate endocytosis. This signaling circuit is also involved in the feedback regulation of PIN-FORMED 1 (PIN1) and PIN2 auxin transporters activity (via its constitutive endocytosis) and corresponding auxin transport-mediated processes, including root gravitropism and leave vascular tissue patterning. Our findings suggest that the signaling module auxin–ABP1–ROP6/RIC1–clathrin–PIN1/PIN2 is a shared component of the feedback regulation of auxin transport during both root and aerial development.

PIN-FORMED (PIN) protein-mediated auxin polar transport is critically important for development, pattern formation, and morphogenesis in plants. Auxin has been implicated in the regulation of polar auxin transport by inhibiting PIN endocytosis [1 and 2], but how auxin regulates this process is poorly understood. Our genetic screen identified the Arabidopsis SPIKE1 (SPK1) gene whose loss-of-function mutations increased lateral root density and retarded gravitropic responses, as do pin2 knockout mutations [3]. SPK1 belongs to the conserved DHR2-Dock family of Rho guanine nucleotide exchange factors [ 4, 5 and 6]. The spk1 mutations induced PIN2 internalization that was not suppressed by auxin, as did the loss-of-function mutations for Rho-like GTPase from Plants 6 (ROP6)-GTPase or its effector RIC1. Furthermore, SPK1 was required for auxin induction of ROP6 activation. Our results have established a Rho GTPase-based auxin signaling pathway that maintains PIN2 polar distribution to the plasma membrane via inhibition of its internalization in Arabidopsis roots. Our findings provide new insights into signaling mechanisms that underlie the regulation of the dynamic trafficking of PINs required for long-distance auxin transport and that link auxin signaling to PIN-mediated pattern formation and morphogenesis.

A study in leaf epidermal pavement cells reveals that auxin activation of a Rho-like GTPase from plants induces inhibition of endocytosis through the clathrin-mediated pathway by regulating the accumulation of cortical F-actin., Cell polarization via asymmetrical distribution of structures or molecules is essential for diverse cellular functions and development of organisms, but how polarity is developmentally controlled has been poorly understood. In plants, the asymmetrical distribution of the PIN-FORMED (PIN) proteins involved in the cellular efflux of the quintessential phytohormone auxin plays a central role in developmental patterning, morphogenesis, and differential growth. Recently we showed that auxin promotes cell interdigitation by activating the Rho family ROP GTPases in leaf epidermal pavement cells. Here we found that auxin activation of the ROP2 signaling pathway regulates the asymmetric distribution of PIN1 by inhibiting its endocytosis. ROP2 inhibits PIN1 endocytosis via the accumulation of cortical actin microfilaments induced by the ROP2 effector protein RIC4. Our findings suggest a link between the developmental auxin signal and polar PIN1 distribution via Rho-dependent cytoskeletal reorganization and reveal the conservation of a design principle for cell polarization that is based on Rho GTPase-mediated inhibition of endocytosis., Author Summary Formation of cell polarity is a process of distributing cellular structures or molecules in an asymmetric manner. This process plays an important role in the generation of diverse cell forms and types. In plants, the quintessential hormone auxin is important for diverse physiological functions, including growth and development of cells and organs. To perform these functions, auxin must be transported and localized to specific regions within the plant. This is partially mediated by polar distribution of the PIN-FORMED (PIN) auxin efflux transporters, which transport auxin outside of the cell and allow for the directional short- and long-distance transport of auxin throughout plant tissues and organs. Although auxin itself has been implicated as a signal to regulate PIN polar distribution, how auxin does so remains to be elucidated. We previously showed that auxin promotes the generation of “puzzle-piece” polarity in leaf epidermal pavement cells, which contain interdigitated lobes and indentations, by activating the ROP (Rho-like GTPases from plants) members of the conserved Rho family of small GTPases. Here, we find that auxin-dependent local activation of ROP2 in the lobe region inhibits PIN1 internalization into the endosomal compartments (or endocytosis), leaving higher levels of PIN1 polar distribution in the lobe region. PIN1 internalization is inhibited by altering the actin cytoskeleton through the ROP2 effector protein RIC4, a protein involved in cytoskeletal remodeling. On the basis of our findings, we propose that the Rho GTPase-mediated inhibition of endocytosis of PIN1 provides a self-organizing mechanism for the polar PIN1 distribution. Rho GTPase-based inhibition of endocytosis is also important for the formation of cell polarity in animal cells. Thus, we conclude that Rho GTPase signaling to inhibit endocytosis is a common mechanism for cell polarization in multicellular organisms.

Within a multicellular tissue cells may coordinately form a singular or multiple polar axes, but it is unclear whether a common mechanism governs different types of polar axis formation. The phosphorylation status of PIN proteins, which is directly affected by the PINOID (PID) protein kinase and the PP2A protein phosphatase, is known to regulate the apical-basal polarity of PIN localization in bipolar cells of roots and shoot apices. Here, we provide evidence that the phosphorylation status-mediated PIN polarity switch is widely used to modulate cellular processes in Arabidopsis including multipolar pavement cells (PC) with interdigitated lobes and indentations. The degree of PC interdigitation was greatly reduced either when the FYPP1 gene, which encodes a PP2A called phytochrome-associated serine/threonine protein phosphatase, was knocked out or when the PID gene was overexpressed (35S::PID). These genetic modifications caused PIN1 localization to switch from lobe to indentation regions. The PP2A and PID mediated switching of PIN1 localization is strikingly similar to their regulation of the apical-basal polarity switch of PIN proteins in other cells. Our findings suggest a common mechanism for the regulation of PIN1 polarity formation, a fundamental cellular process that is crucial for pattern formation both at the tissue/organ and cellular levels.

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