Tatsuaki Goh

Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions

Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication—a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms. Polar PIN localization determines the direction of intercellular auxin flow, yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosis-dependent mechanism of PIN polarity generation and analyse its developmental implications. Real-time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.

Lateral root development in Arabidopsis: fifty shades of auxin

The developmental plasticity of the root system represents a key adaptive trait enabling plants to cope with abiotic stresses such as drought and is therefore important in the current context of global changes. Root branching through lateral root formation is an important component of the adaptability of the root system to its environment. Our understanding of the mechanisms controlling lateral root development has progressed tremendously in recent years through research in the model plant Arabidopsis thaliana (Arabidopsis). These studies have revealed that the phytohormone auxin acts as a common integrator to many endogenous and environmental signals regulating lateral root formation. Here, we review what has been learnt about the myriad roles of auxin during lateral root formation in Arabidopsis.

The establishment of asymmetry in Arabidopsis lateral root founder cells is regulated by LBD16/ASL18 and related LBD/ASL proteins

In most dicot plants, lateral root (LR) formation, which is important for the construction of the plant root system, is initiated from coordinated asymmetric cell divisions (ACD) of the primed LR founder cells in the xylem pole pericycle (XPP) of the existing roots. In Arabidopsis thaliana, two AUXIN RESPONSE FACTORs (ARFs), ARF7 and ARF19, positively regulate LR formation through activation of the plant-specific transcriptional regulators LATERAL ORGAN BOUNDARIES-DOMAIN 16/ASYMMETRIC LEAVES2-LIKE 18 (LBD16/ASL18) and the other related LBD/ASL genes. The exact biological role of these LBD/ASLs in LR formation is still unknown. Here, we demonstrate that LBD16/ASL18 is specifically expressed in the LR founder cells adjacent to the XPP before the first ACD and that it functions redundantly with the other auxin-inducible LBD/ASLs in LR initiation. The spatiotemporal expression of LBD16/ASL18 during LR initiation is dependent on the SOLITARY-ROOT (SLR)/IAA14-ARF7-ARF19 auxin signaling module. In addition, XPP-specific expression of LBD16/ASL18 in arf7 arf19 induced cell divisions at XPP, thereby restoring the LR phenotype. We also demonstrate that expression of LBD16-SRDX, a dominant repressor of LBD16/ASL18 and its related LBD/ASLs, does not interfere in the specification of LR founder cells with local activation of the auxin response, but it blocks the polar nuclear migration in LR founder cells before ACD, thereby blocking the subsequent LR initiation. Taken together, these results indicate that the localized activity of LBD16/ASL18 and its related LBD/ASLs is involved in the symmetry breaking of LR founder cells for LR initiation, a key step for constructing the plant root system.

VPS9a, the Common Activator for Two Distinct Types of Rab5 GTPases, Is Essential for the Development of Arabidopsis thaliana

Rab5, a subfamily of Rab GTPases, regulates a variety of endosomal functions as a molecular switch. Arabidopsis thaliana has two different types of Rab5-member GTPases: conventional type, ARA7 and RHA1, and a plant-specific type, ARA6. We found that only one guanine nucleotide exchange factor (GEF), named VPS9a, can activate all Rab5 members to GTP-bound forms in vitro in spite of their diverged structures. In the vps9a-1 mutant, whose GEF activity is completely lost, embryogenesis was arrested at the torpedo stage. Green fluorescent protein (GFP)–ARA7 and ARA6-GFP were diffused in cytosol like GDP-fixed mutants of Rab5 in vps9a-1, indicating that both types of GTPase are regulated by VPS9a. In the leaky vps9a-2 mutant, elongation of the primary root was severely affected. Overexpression of the GTP-fixed form of ARA7 suppressed the vps9a-2 mutation, but overexpression of ARA6 had no apparent effects. These results indicate that the two types of plant Rab5 members are functionally differentiated, even though they are regulated by the same activator, VPS9a.

Dynamic Aspects of Ion Accumulation by Vesicle Traffic Under Salt Stress in Arabidopsis

The intracellular membrane dynamics of Arabidopsis cells under high salt treatment were investigated. When Arabidopsis was treated with high levels of NaCl in hydroponic culture, root tip cells showed rapid changes in the vacuolar volume, a decrease in the number of small acid compartments, active movement of vesicles and accumulation of Na(+) both in the central vacuole and in the vesicles around the main vacuole observed with the Na(+)-dependent fluorescence of Sodium Green. Detailed observation of Arabidopsis suspension-cultured cells under high salt treatment showed a similar pattern of response to that observed in root tip cells. Immunostaining of suspension-cultured cells with antibodies against AtNHX1 clearly showed the occurrence of dotted fluorescence in the cytoplasm only under salt treatment. We also confirmed the existence of AtNHX1 in the vacuolar membrane isolated from suspension-cultured cells with immunofluorescence. Knockout of the vacuolar Q(a)-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein VAM3/SYP22 caused an increase in salt tolerance. In mutant plants, the distribution of Na(+) between roots and shoots differed from that of wild-type plants, with Na(+) accumulating more in roots and less in the shoots of the mutant plants. The role of vesicle traffic under salt stress is discussed.

Multiple AUX/IAA–ARF modules regulate lateral root formation: the role of Arabidopsis SHY2/IAA3-mediated auxin signalling

In Arabidopsis thaliana, lateral root (LR) formation is regulated by multiple auxin/indole-3-acetic acid (Aux/IAA)–AUXIN RESPONSE FACTOR (ARF) modules: (i) the IAA28–ARFs module regulates LR founder cell specification; (ii) the SOLITARY-ROOT (SLR)/IAA14–ARF7–ARF19 module regulates nuclear migration and asymmetric cell divisions of the LR founder cells for LR initiation; and (iii) the BODENLOS/IAA12–MONOPTEROS/ARF5 module also regulates LR initiation and organogenesis. The number of Aux/IAA–ARF modules involved in LR formation remains unknown. In this study, we isolated the shy2-101 mutant, a gain-of-function allele of short hypocotyl2/suppressor of hy2 (shy2)/iaa3 in the Columbia accession. We demonstrated that the shy2-101 mutation not only strongly inhibits LR primordium development and emergence but also significantly increases the number of LR initiation sites with the activation of LATERAL ORGAN BOUNDARIES-DOMAIN16/ASYMMETRIC LEAVES2-LIKE18, a target gene of the SLR/IAA14–ARF7–ARF19 module. Genetic analysis revealed that enhanced LR initiation in shy2-101 depended on the SLR/IAA14–ARF7–ARF19 module. We also showed that the shy2 roots contain higher levels of endogenous IAA. These observations indicate that the SHY2/IAA3–ARF-signalling module regulates not only LR primordium development and emergence after SLR/IAA14–ARF7–ARF19 module-dependent LR initiation but also inhibits LR initiation by affecting auxin homeostasis, suggesting that multiple Aux/IAA–ARF modules cooperatively regulate the developmental steps during LR formation.

Plant vacuolar trafficking occurs through distinctly regulated pathways.

The multifunctional vacuole is the largest organelle in plant cells, and many proteins are transported to and stored in this organelle; thus, the vacuole has great physiological and agronomical importance. However, the molecular mechanism and regulation of plant vacuolar traffic remain largely unknown. In this study, we demonstrate that multiple vacuolar trafficking pathways operate in plants. RAB5 and RAB7 are evolutionarily conserved subfamilies of Rab GTPase, whose animal and yeast counterparts regulate vacuolar/endosomal trafficking in a sequential manner. Functional analyses of a putative activating complex for RAB7 indicated that this complex is responsible for maturation from RAB5- to RAB7-positive endosomes in plant cells. Moreover, these machinery components are recruited to a more complex trafficking network. Mutations in RAB5 and RAB7 conferred counteracting effects on the vti11 mutant. Furthermore, impairment of RAB5- and RAB7-dependent pathways differentially affected the transport of distinctive cargos. These results indicate that plants have developed a complex vacuolar transport system distinct from that of nonplant systems by assigning evolutionarily conserved machinery to unique trafficking pathways. These pathways provide a fundamental basis for plant development at the cellular and higher-ordered levels.

The circadian clock rephases during lateral root organ initiation in Arabidopsis thaliana

The endogenous circadian clock enables organisms to adapt their growth and development to environmental changes. Here we describe how the circadian clock is employed to coordinate responses to the key signal auxin during lateral root (LR) emergence. In the model plant, Arabidopsis thaliana, LRs originate from a group of stem cells deep within the root, necessitating that new organs emerge through overlying root tissues. We report that the circadian clock is rephased during LR development. Metabolite and transcript profiling revealed that the circadian clock controls the levels of auxin and auxin-related genes including the auxin response repressor IAA14 and auxin oxidase AtDAO2. Plants lacking or overexpressing core clock components exhibit LR emergence defects. We conclude that the circadian clock acts to gate auxin signalling during LR development to facilitate organ emergence.

Inference of the Arabidopsis Lateral Root Gene Regulatory Network Suggests a Bifurcation Mechanism That Defines Primordia Flanking and Central Zones

A new correlation algorithm infers the Arabidopsis lateral root gene regulatory network from time-course transcriptomic data and provides mechanistic insight into primordia patterning. A large number of genes involved in lateral root (LR) organogenesis have been identified over the last decade using forward and reverse genetic approaches in Arabidopsis thaliana. Nevertheless, how these genes interact to form a LR regulatory network largely remains to be elucidated. In this study, we developed a time-delay correlation algorithm (TDCor) to infer the gene regulatory network (GRN) controlling LR primordium initiation and patterning in Arabidopsis from a time-series transcriptomic data set. The predicted network topology links the very early-activated genes involved in LR initiation to later expressed cell identity markers through a multistep genetic cascade exhibiting both positive and negative feedback loops. The predictions were tested for the key transcriptional regulator AUXIN RESPONSE FACTOR7 node, and over 70% of its targets were validated experimentally. Intriguingly, the predicted GRN revealed a mutual inhibition between the ARF7 and ARF5 modules that would control an early bifurcation between two cell fates. Analyses of the expression pattern of ARF7 and ARF5 targets suggest that this patterning mechanism controls flanking and central zone specification in Arabidopsis LR primordia.

A role for LATERAL ORGAN BOUNDARIES‐DOMAIN 16 during the interaction Arabidopsis–Meloidogyne spp. provides a molecular link between lateral root and root‐knot nematode feeding site development

Plant endoparasitic nematodes induce the formation of their feeding cells by injecting effectors from the esophageal glands into root cells. Although vascular cylinder cells seem to be involved in the formation of root-knot nematode (RKN) feeding structures, molecular evidence is scarce. We address the role during gall development of LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16), a key component of the auxin pathway leading to the divisions in the xylem pole pericycle (XPP) for lateral root (LR) formation. Arabidopsis T-DNA tagged J0192 and J0121 XPP marker lines, LBD16 and DR5::GUS promoter lines, and isolated J0192 protoplasts were assayed for nematode-dependent gene expression. Infection tests in LBD16 knock-out lines were used for functional analysis. J0192 and J0121 lines were activated in early developing galls and giant cells (GCs), resembling the pattern of the G2/M-transition specific ProC yc B 1;1 :CycB1;1(NT)-GUS line. LBD16 was regulated by auxins in galls as in LRs, and induced by RKN secretions. LBD16 loss of function mutants and a transgenic line with defective XPP cells showed a significantly reduced infection rate. The results show that genes expressed in the dividing XPP, particularly LBD16, are important for gall formation, as they are for LR development.