ROP Interactive Partners are Involved in the Control of Cell Division Patterns in Arabidopsis Leaves.

Animal Rho GTP-binding proteins and their plant counterparts, Rho of plants (ROPs), regulate cell polarity, but they do so through different effector proteins. A class of ROP effectors, interactor of constitutive active ROPs (ICRs)/ROP interactive partners (RIPs), has been implicated in diverse biological processes; however, there are limited analyses of RIP loss-of-function mutants. Here, we report an analysis of the functions of the Arabidopsis thaliana RIPs in the leaf epidermis. Green Fluorescent Protein (GFP) fusion proteins of all the RIPs colocalized to cortical microtubules. RIP1, RIP3 and RIP4, but not RIP2 and RIP5, colocalized with the preprophase band (PPB), spindles and phragmoplasts. RIP2 and RIP5 did not colocalize with the PPB, spindles or phragmoplasts even when they were expressed under a promoter active in proliferative cells, indicating that there are differences among RIP protein properties. The overexpression of RIP1 or RIP4 resulted in the fragmentation of cortical microtubules, and the rip1 2 3 4 5 quintuple mutant showed increased growth rate of microtubules at their plus ends compared with the wild type. The rip1 2 3 4 5 mutant leaves and petals were narrow, which was explained by the decreased cell number along the transverse axis compared with that of the wild type. The rip1 2 3 4 5 mutant leaf epidermis possessed fewer PPBs oriented close to the long axis of the leaf compared with wild type, indicating the involvement of RIPs in cell division plane regulation and leaf shape determination.

Pericycle cell division competence underlies various developmental programs.

Pericycle cells possess proliferative activity long after leaving the root apical meristem. Depending on the developmental stage and external stimuli, pericycle cell division leads to the production of lateral roots, vascular cambium and periderm, and callus. Therefore, pericycle cell division competence underlies root branching and secondary growth, as well as plant regeneration capacity. In this review, we first briefly present an overview of the molecular pathways of the four developmental programs originated, exclusively or partly, from pericycle cells. Then, we provide a review of up-to-date knowledge in the mechanisms determining pericycle cells' competence to undergo cell division. Furthermore, we discuss directions of future research to further our understanding of the pericycle's characteristics and functions.

A Dof-CLE circuit controls phloem organization.

The phloem consists of sieve elements (SEs) and companion cells (CCs). Here we show that Dof-class transcription factors preferentially expressed in the phloem (phloem-Dofs) are not only necessary and sufficient for SE and CC differentiation, but also induce negative regulators of phloem development, CLAVATA3/EMBRYO SURROUNDING REGION-RELATED25 (CLE25), CLE26 and CLE45 secretory peptides. CLEs were perceived by BARELY ANY MERISTEM (BAM)-class receptors and CLAVATA3 INSENSITIVE RECEPTOR KINASE (CIK) co-receptors, and post-transcriptionally decreased phloem-Dof proteins and repressed SE and CC formation. Multiple mutations in CLE-, BAM- or CIK-class genes caused ectopic formation of SEs and CCs, producing an SE/CC cluster at each phloem region. We propose that while phloem-Dofs induce phloem cell formation, they inhibit excess phloem cell formation by inducing CLEs. Normal-positioned SE and CC precursor cells appear to overcome the effect of CLEs by reinforcing the production of phloem-Dofs through a positive feedback transcriptional regulation.

Two types of bHLH transcription factor determine the competence of the pericycle for lateral root initiation.

The molecular basis of the competence of the pericycle cell to initiate lateral root primordium formation is totally unknown. Here, we report that in Arabidopsis, two types of basic helix-loop-helix (bHLH) transcription factors, named PERICYCLE FACTOR TYPE-A (PFA) proteins and PERICYCLE FACTOR TYPE-B (PFB) proteins, govern the competence of pericycle cells to initiate lateral root primordium formation. Overexpression of PFA genes confers hallmark pericycle characteristics, including specific marker gene expression and auxin-induced cell division, and multiple loss-of-function mutations in PFA genes or the repression of PFB target genes results in the loss of this specific pericycle function. PFA and PFB proteins physically interact and are under mutual- and self-regulation, forming a positive feedback loop. This study unveils the transcriptional regulatory system that determines pericycle participation in lateral root initiation.

Early Endosomal Trafficking Component BEN2/VPS45 Plays a Crucial Role in Internal Tissues in Regulating Root Growth and Meristem Size in Arabidopsis.

Polar auxin transport is involved in multiple aspects of plant development, including root growth, lateral root branching, embryogenesis, and vasculature development. PIN-FORMED (PIN) auxin efflux proteins exhibit asymmetric distribution at the plasma membrane (PM) and collectively play pivotal roles in generating local auxin accumulation, which underlies various auxin-dependent developmental processes. In previous research, it has been revealed that endosomal trafficking components BEN1/BIG5 (ARF GEF) and BEN2/VPS45 (Sec1/Munc 18 protein) function in intracellular trafficking of PIN proteins in Arabidopsis. Mutations in both BEN1 and BEN2 resulted in defects in polar PIN localization, auxin response gradients, and in root architecture. In this study, we have attempted to gain insight into the developmental roles of these trafficking components. We showed that while genetic or pharmacological disturbances of auxin distribution reduced dividing cells in the root tips and resulted in reduced root growth, the same manipulations had only moderate impact on ben1; ben2 double mutants. In addition, we established transgenic lines in which BEN2/VPS45 is expressed under control of tissue-specific promoters and demonstrated that BEN2/VPS45 regulates the intracellular traffic of PIN proteins in cell-autonomous manner, at least in stele and epidermal cells. Furthermore, BEN2/VPS45 rescued the root architecture defects when expressed in internal tissues of ben1; ben2 double mutants. These results corroborate the roles of the endosomal trafficking component BEN2/VPS45 in regulation of auxin-dependent developmental processes, and suggest that BEN2/VPS45 is required for sustainable root growth, most likely through regulation of tip-ward auxin transport through the internal tissues of root.

Author Correction: The CLE9/10 secretory peptide regulates stomatal and vascular development through distinct receptors.

In the version of this Article originally published, the authors incorrectly stated that the work was supported by Innovative Areas grant number 25003006; the correct number is 25113006. This statement has now been amended in all online versions of the Article.

The CLE9/10 secretory peptide regulates stomatal and vascular development through distinct receptors.

The frequency and orientation of cell division are regulated by intercellular signalling molecules; however, tissue-specific regulatory systems for cell divisions are only partially understood. Here, we report that the peptide hormone CLAVATA3/ESR-RELATED 9/10 (CLE9/10) regulates two different developmental processes, stomatal lineage development and xylem development, through two distinct receptor systems in Arabidopsis thaliana. We show that the receptor kinase HAESA-LIKE 1 (HSL1) is a CLE9/10 receptor that regulates stomatal lineage cell division, and BARELY NO MERISTEM (BAM) class receptor kinases are CLE9/10 receptors that regulate periclinal cell division of xylem precursor cells. Both HSL1 and BAM1 bind to CLE9/10, but only HSL1 recruits SOMATIC EMBRYOGENESIS RECEPTOR KINASES as co-receptors in the presence of CLE9/10, suggesting different signalling modes for these receptor systems.

Cytokinin signalling regulates organ identity via the AHK4 receptor in Arabidopsis.

Mutual interactions of the phytohormones, cytokinins and auxin determine root or shoot identity during postembryonic de novo organogenesis in plants. However, our understanding of the role of hormonal metabolism and perception during early stages of cell fate reprogramming is still elusive. Here we show that auxin activates root formation, whereas cytokinins mediate early loss of the root identity, primordia disorganisation and initiation of shoot development. Exogenous and endogenous cytokinins influence the initiation of newly formed organs, as well as the pace of organ development. The process of de novo shoot apical meristem establishment is accompanied by accumulation of endogenous cytokinins, differential regulation of genes for individual cytokinin receptors, strong activation of AHK4-mediated signalling and induction of the shoot-specific homeodomain regulator WUSCHEL. The last is associated with upregulation of isopentenyladenine-type cytokinins, revealing higher shoot-forming potential when compared with trans-zeatin. Moreover, AHK4-controlled cytokinin signalling negatively regulates the root stem cell organiser WUSCHEL RELATED HOMEOBOX 5 in the root quiescent centre. We propose an important role for endogenous cytokinin biosynthesis and AHK4-mediated cytokinin signalling in the control of de novo-induced organ identity.

Asymmetric Auxin Distribution is Not Required to Establish Root Phototropism in Arabidopsis.

An asymmetric auxin distribution pattern is assumed to underlie the tropic responses of seed plants. It is unclear, however, whether this pattern is required for root negative phototropism. We here demonstrate that asymmetric auxin distribution is not required to establish root phototropism in Arabidopsis. Our detailed analyses of auxin reporter genes indicate that auxin accumulates on the irradiated side of roots in response to an incidental gravitropic stimulus caused by phototropic bending. Further, an agravitropic mutant showed a suppression of this accumulation with an enhancement of the phototropic response. In this context, our pharmacological and genetic analyses revealed that both polar auxin transport and auxin biosynthesis are critical for the establishment of root gravitropism, but not for root phototropism, and that defects in these processes actually enhance phototropic responses in roots. The auxin response factor double mutant arf7 arf19 and the auxin receptor mutant tir1 showed a slight reduction in phototropic curvatures in roots, suggesting that the transcriptional regulation by some specific ARF proteins and their regulators is at least partly involved in root phototropism. However, the auxin antagonist PEO-IAA [α-(phenylethyl-2-one)-indole-3-acetic acid] suppressed root gravitropism and enhanced root phototropism, suggesting that the TIR1/AFB auxin receptors and ARF transcriptional factors play minor roles in root phototropism. Taken together, we conclude from our current data that the phototropic response in Arabidopsis roots is induced by an unknown mechanism that does not require asymmetric auxin distribution and that the Cholodny-Went hypothesis probably does not apply to root phototropism.

BEN3/BIG2 ARF GEF is Involved in Brefeldin A-Sensitive Trafficking at the trans-Golgi Network/Early Endosome in Arabidopsis thaliana.

Membrane traffic at the trans-Golgi network (TGN) is crucial for correctly distributing various membrane proteins to their destination. Polarly localized auxin efflux proteins, including PIN-FORMED1 (PIN1), are dynamically transported between the endosomes and the plasma membrane (PM) in the plant cells. The intracellular trafficking of PIN1 protein is sensitive to the fungal toxin brefeldin A (BFA), which is known to inhibit guanine nucleotide exchange factors for ADP ribosylation factors (ARF GEFs) such as GNOM. However, the molecular details of the BFA-sensitive trafficking pathway have not been fully revealed. In a previous study, we identified an Arabidopsis mutant BFA-visualized endocytic trafficking defective 3 (ben3) which exhibited reduced sensitivity to BFA in terms of BFA-induced intracellular PIN1 agglomeration. Here, we show that BEN3 encodes a member of BIG family ARF GEFs, BIG2. BEN3/BIG2 tagged with fluorescent proteins co-localized with markers for the TGN/early endosome (EE). Inspection of conditionally induced de novo synthesized PIN1 confirmed that its secretion to the PM is BFA sensitive, and established BEN3/BIG2 as a crucial component of this BFA action at the level of the TGN/EE. Furthermore, ben3 mutation alleviated BFA-induced agglomeration of another TGN-localized ARF GEF, BEN1/MIN7. Taken together, our results suggest that BEN3/BIG2 is an ARF GEF component, which confers BFA sensitivity to the TGN/EE in Arabidopsis.

Divergent expression of cytokinin biosynthesis, signaling and catabolism genes underlying differences in feeding sites induced by cyst and root-knot nematodes.

Cyst and root-knot nematodes are obligate parasites of economic importance with a remarkable ability to reprogram root cells into unique metabolically active feeding sites. Previous studies have suggested a role for cytokinin in feeding site formation induced by these two types of nematodes, but the mechanistic details have not yet been described. Using Arabidopsis as a host plant species, we conducted a comparative analysis of cytokinin genes in response to the beet cyst nematode (BCN), Heterodera schachtii, and the root-knot nematode (RKN), Meloidogyne incognita. We identified distinct differences in the expression of cytokinin biosynthesis, catabolism and signaling genes in response to infection by BCN and RKN, suggesting differential manipulation of the cytokinin pathway by these two nematode species. Furthermore, we evaluated Arabidopsis histidine kinase receptor mutant lines ahk2/3, ahk2/4 and ahk3/4 in response to RKN infection. Similar to our previous studies with BCN, these lines were significantly less susceptible to RKN without compromising nematode penetration, suggesting a requirement of cytokinin signaling in RKN feeding site formation. Moreover, an analysis of ahk double mutants using CycB1;1:GUS/ahk introgressed lines revealed contrasting differences in the cytokinin receptors mediating cell cycle activation in feeding sites induced by BCN and RKN.

A parasitic nematode releases cytokinin that controls cell division and orchestrates feeding site formation in host plants.

Sedentary plant-parasitic cyst nematodes are biotrophs that cause significant losses in agriculture. Parasitism is based on modifications of host root cells that lead to the formation of a hypermetabolic feeding site (a syncytium) from which nematodes withdraw nutrients. The host cell cycle is activated in an initial cell selected by the nematode for feeding, followed by activation of neighboring cells and subsequent expansion of feeding site through fusion of hundreds of cells. It is generally assumed that nematodes manipulate production and signaling of the plant hormone cytokinin to activate cell division. In fact, nematodes have been shown to produce cytokinin in vitro; however, whether the hormone is secreted into host plants and plays a role in parasitism remained unknown. Here, we analyzed the spatiotemporal activation of cytokinin signaling during interaction between the cyst nematode, Heterodera schachtii, and Arabidopsis using cytokinin-responsive promoter:reporter lines. Our results showed that cytokinin signaling is activated not only in the syncytium but also in neighboring cells to be incorporated into syncytium. An analysis of nematode infection on mutants that are deficient in cytokinin or cytokinin signaling revealed a significant decrease in susceptibility of these plants to nematodes. Further, we identified a cytokinin-synthesizing isopentenyltransferase gene in H. schachtii and show that silencing of this gene in nematodes leads to a significant decrease in virulence due to a reduced expansion of feeding sites. Our findings demonstrate the ability of a plant-parasitic nematode to synthesize a functional plant hormone to manipulate the host system and establish a long-term parasitic interaction.

AINTEGUMENTA and the D-type cyclin CYCD3;1 regulate root secondary growth and respond to cytokinins.

Higher plant vasculature is characterized by two distinct developmental phases. Initially, a well-defined radial primary pattern is established. In eudicots, this is followed by secondary growth, which involves development of the cambium and is required for efficient water and nutrient transport and wood formation. Regulation of secondary growth involves several phytohormones, and cytokinins have been implicated as key players, particularly in the activation of cell proliferation, but the molecular mechanisms mediating this hormonal control remain unknown. Here we show that the genes encoding the transcription factor AINTEGUMENTA (ANT) and the D-type cyclin CYCD3;1 are expressed in the vascular cambium of Arabidopsis roots, respond to cytokinins and are both required for proper root secondary thickening. Cytokinin regulation of ANT and CYCD3 also occurs during secondary thickening of poplar stems, suggesting this represents a conserved regulatory mechanism.

Distinct Characteristics of Indole-3-Acetic Acid and Phenylacetic Acid, Two Common Auxins in Plants.

The phytohormone auxin plays a central role in many aspects of plant growth and development. IAA is the most studied natural auxin that possesses the property of polar transport in plants. Phenylacetic acid (PAA) has also been recognized as a natural auxin for >40 years, but its role in plant growth and development remains unclear. In this study, we show that IAA and PAA have overlapping regulatory roles but distinct transport characteristics as auxins in plants. PAA is widely distributed in vascular and non-vascular plants. Although the biological activities of PAA are lower than those of IAA, the endogenous levels of PAA are much higher than those of IAA in various plant tissues in Arabidopsis. PAA and IAA can regulate the same set of auxin-responsive genes through the TIR1/AFB pathway in Arabidopsis. IAA actively forms concentration gradients in maize coleoptiles in response to gravitropic stimulation, whereas PAA does not, indicating that PAA is not actively transported in a polar manner. The induction of the YUCCA (YUC) genes increases PAA metabolite levels in Arabidopsis, indicating that YUC flavin-containing monooxygenases may play a role in PAA biosynthesis. Our results provide new insights into the regulation of plant growth and development by different types of auxins.

Cytokinin is required for escape but not release from auxin mediated apical dominance.

Auxin produced by an active primary shoot apex is transported down the main stem and inhibits the growth of the axillary buds below it, contributing to apical dominance. Here we use Arabidopsis thaliana cytokinin (CK) biosynthetic and signalling mutants to probe the role of CK in this process. It is well established that bud outgrowth is promoted by CK, and that CK synthesis is inhibited by auxin, leading to the hypothesis that release from apical dominance relies on an increased supply of CK to buds. Our data confirm that decapitation induces the expression of at least one ISOPENTENYLTRANSFERASE (IPT) CK biosynthetic gene in the stem. We further show that transcript abundance of a clade of the CK-responsive type-A Arabidopsis response regulator (ARR) genes increases in buds following CK supply, and that, contrary to their typical action as inhibitors of CK signalling, these genes are required for CK-mediated bud activation. However, analysis of the relevant arr and ipt multiple mutants demonstrates that defects in bud CK response do not affect auxin-mediated bud inhibition, and increased IPT transcript levels are not needed for bud release following decapitation. Instead, our data suggest that CK acts to overcome auxin-mediated bud inhibition, allowing buds to escape apical dominance under favourable conditions, such as high nitrate availability.

Pathways involved in vanadate-induced root hair formation in Arabidopsis.

Root hair formation is controlled by environmental signals. We found significantly increased Arabidopsis root hair density and length in response to low-dose vanadate (V). Reactive oxygen species (ROS) production was induced with V treatment. We investigated the possible role of NADPH oxidase in altering root system architecture induced by V by using diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, and an NADPH oxidase mutant (rhd2/AtrbohC). NADPH oxidase was involved in root hair elongation induced by V. As well, ethylene receptor (ETR1) and ROOT HAIR DEFECTIVE (RHD6) participated in inducing root hair formation induced by V. Furthermore, the kinase inhibitors, genistein (tyrosine kinase inhibitor) and K252a (ser/thr kinase inhibitor), and a phosphatase inhibitor, cantharidin (ser/thr phosphatase inhibitor), suppressed root hair formation induced by V. To elucidate the regulation of gene expression in response to V, we investigated transcriptional changes in roots by microarray assay. Exposure to V triggered changes in transcript levels of genes related to cell wall formation, ROS activity and signaling. Several genes involved in root hair formation were also regulated.

Arabidopsis reduces growth under osmotic stress by decreasing SPEECHLESS protein.

Plants, which are sessile unlike most animals, have evolved a system to reduce growth under stress; however, the molecular mechanisms of this stress response are not well known. During programmed development, a fraction of the leaf epidermal precursor cells become meristemoid mother cells (MMCs), which are stem cells that produce both stomatal guard cells and epidermal pavement cells. Here we report that Arabidopsis plants, in response to osmotic stress, post-transcriptionally decrease the protein level of SPEECHLESS, the transcription factor promoting MMC identity, through the action of a mitogen-activated protein kinase (MAPK) cascade. The growth reduction under osmotic stress was lessened by inhibition of the MAPK cascade or by a mutation that disrupted the MAPK target amino acids in SPEECHLESS, indicating that Arabidopsis reduces growth under stress by integrating the osmotic stress signal into the MAPK-SPEECHLESS core developmental pathway.

Auxin sensitivities of all Arabidopsis Aux/IAAs for degradation in the presence of every TIR1/AFB.

Auxin plays a key role in regulation of almost all processes of plant growth and development. Different physiological processes are regulated by different ranges of auxin concentrations; however, the underlying mechanisms creating these differences are largely unknown. The first step of auxin signaling is auxin-dependent interaction of an auxin receptor with transcriptional co-repressors (Aux/IAA), which leads to Aux/IAA degradation. Arabidopsis has six homologous auxin receptors (TIR1 and five AFBs), 29 Aux/IAA proteins and two types of active auxins, IAA and phenylacetic acid (PAA). Therefore, a large number of possible combinations between these three factors may contribute to the creation of complex auxin responses. Using a yeast heterologous reconstitution system, we investigated auxin-dependent degradation of all Arabidopsis Aux/IAAs in combination with every TIR or AFB receptor component. We found that TIR1 and AFB2 were effective in mediating Aux/IAA degradation. We confirmed that the Aux/IAA domain II, which binds TIR1, is essential for degradation. IAA and other natural auxins, 4-chloroindole-3-acetic acid (4-Cl-IAA) and PAA, induced Aux/IAA degradation; and IAA and 4-Cl-IAA had higher activity than PAA. Effective auxin concentrations for Aux/IAA degradation depended on both Aux/IAAs and TIR1 or AFB2 receptors, which is consistent with the Aux/IAA-TIR1/AFB co-receptor concept.

BEX1/ARF1A1C is required for BFA-sensitive recycling of PIN auxin transporters and auxin-mediated development in Arabidopsis.

Correct positioning of membrane proteins is an essential process in eukaryotic organisms. The plant hormone auxin is distributed through intercellular transport and triggers various cellular responses. Auxin transporters of the PIN-FORMED (PIN) family localize asymmetrically at the plasma membrane (PM) and mediate the directional transport of auxin between cells. A fungal toxin, brefeldin A (BFA), inhibits a subset of guanine nucleotide exchange factors for ADP-ribosylation factor small GTPases (ARF GEFs) including GNOM, which plays a major role in localization of PIN1 predominantly to the basal side of the PM. The Arabidopsis genome encodes 19 ARF-related putative GTPases. However, ARF components involved in PIN1 localization have been genetically poorly defined. Using a fluorescence imaging-based forward genetic approach, we identified an Arabidopsis mutant, bfa-visualized exocytic trafficking defective1 (bex1), in which PM localization of PIN1-green fluorescent protein (GFP) as well as development is hypersensitive to BFA. We found that in bex1 a member of the ARF1 gene family, ARF1A1C, was mutated. ARF1A1C localizes to the trans-Golgi network/early endosome and Golgi apparatus, acts synergistically to BEN1/MIN7 ARF GEF and is important for PIN recycling to the PM. Consistent with the developmental importance of PIN proteins, functional interference with ARF1 resulted in an impaired auxin response gradient and various developmental defects including embryonic patterning defects and growth arrest. Our results show that ARF1A1C is essential for recycling of PIN auxin transporters and for various auxin-dependent developmental processes.

Cell polarity and patterning by PIN trafficking through early endosomal compartments in Arabidopsis thaliana.

PIN-FORMED (PIN) proteins localize asymmetrically at the plasma membrane and mediate intercellular polar transport of the plant hormone auxin that is crucial for a multitude of developmental processes in plants. PIN localization is under extensive control by environmental or developmental cues, but mechanisms regulating PIN localization are not fully understood. Here we show that early endosomal components ARF GEF BEN1 and newly identified Sec1/Munc18 family protein BEN2 are involved in distinct steps of early endosomal trafficking. BEN1 and BEN2 are collectively required for polar PIN localization, for their dynamic repolarization, and consequently for auxin activity gradient formation and auxin-related developmental processes including embryonic patterning, organogenesis, and vasculature venation patterning. These results show that early endosomal trafficking is crucial for cell polarity and auxin-dependent regulation of plant architecture.