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Organ-specific gene expression datasets that include hundreds to thousands of experiments allow the reconstruction of organ-level gene regulatory networks (GRNs). However, creating such datasets is greatly hampered by the requirements of extensive and tedious manual curation. Here, we trained a supervised classification model that can accurately classify the organ-of-origin for a plant transcriptome. This K-Nearest Neighbor-based multiclass classifier was used to create organ-specific gene expression datasets for the leaf, root, shoot, flower, and seed in Arabidopsis thaliana . A GRN inference approach was used to determine the: i. influential transcription factors (TFs) in each organ and, ii. most influential TFs for specific biological processes in that organ. These genome-wide, organ-delimited GRNs (OD-GRNs), recalled many known regulators of organ development and processes operating in those organs. Importantly, many previously unknown TF regulators were uncovered as potential regulators of these processes. As a proof-of-concept, we focused on experimentally validating the predicted TF regulators of lipid biosynthesis in seeds, an important food and biofuel trait. Of the top 20 predicted TFs, eight are known regulators of seed oil content, e.g., WRI1, LEC1, FUS3. Importantly, we validated our prediction of MybS2, TGA4, SPL12, AGL18, and DiV2 as regulators of seed lipid biosynthesis. We elucidated the molecular mechanism of MybS2 and show that it induces purple acid phosphatase family genes and lipid synthesis genes to enhance seed lipid content. This general approach has the potential to be extended to any species with sufficiently large gene expression datasets to find unique regulators of any trait-of-interest., Competing Interests: Competing interests statement:The authors have applied for a patent based on the results in this manuscript at Purdue University.

AUXIN RESISTANCE4 (AXR4) regulates the trafficking of auxin influx carrier AUXIN1 (AUX1), a plasma-membrane protein that predominantly localizes to the endoplasmic reticulum (ER) in the absence of AXR4. In Arabidopsis (Arabidopsis thaliana), AUX1 is a member of a small multigene family comprising 4 highly conserved genes-AUX1, LIKE-AUX1 (LAX1), LAX2, and LAX3. We report here that LAX2 also requires AXR4 for correct localization to the plasma membrane. AXR4 is a plant-specific protein and contains a weakly conserved α/β hydrolase fold domain that is found in several classes of lipid hydrolases and transferases. We have previously proposed that AXR4 may either act as (i) a post-translational modifying enzyme through its α/β hydrolase fold domain or (ii) an ER accessory protein, which is a special class of ER protein that regulates targeting of their cognate partner proteins. Here, we show that AXR4 is unlikely to act as a post-translational modifying enzyme as mutations in several highly conserved amino acids in the α/β hydrolase fold domain can be tolerated and active site residues are missing. We also show that AUX1 and AXR4 physically interact with each other and that AXR4 reduces aggregation of AUX1 in a dose-dependent fashion. Our results suggest that AXR4 acts as an ER accessory protein. A better understanding of AXR4-mediated trafficking of auxin transporters in crop plants will be crucial for improving root traits (designer roots) for better acquisition of water and nutrients for sustainable and resilient agriculture., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Oxygen is a key signalling component of plant biology, and whilst an oxygen-sensing mechanism was previously described in Arabidopsis thaliana, key features of the associated PLANT CYSTEINE OXIDASE (PCO) N-degron pathway and Group VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factor substrates remain untested or unknown. We demonstrate that ERFVIIs show non-autonomous activation of root hypoxia tolerance and are essential for root development and survival under oxygen limiting conditions in soil. We determine the combined effects of ERFVIIs in controlling gene expression and define genetic and environmental components required for proteasome-dependent oxygen-regulated stability of ERFVIIs through the PCO N-degron pathway. Using a plant extract, unexpected amino-terminal cysteine sulphonic acid oxidation level of ERFVIIs was observed, suggesting a requirement for additional enzymatic activity within the pathway. Our results provide a holistic understanding of the properties, functions and readouts of this oxygen-sensing mechanism defined through its role in modulating ERFVII stability., (© 2023. Springer Nature Limited.)

Plants can retain a memory of previous pathogen infections to mount a more robust defense response during subsequent infections by developing systemic acquired resistance (SAR). However, the mechanism through which plants develop and retain infection memory is not known. Experiments have shown the association of epigenetic modifications of specific defense-related genes with SAR. RSI1/FLD codes for a histone demethylase and is required for the activation of SAR in Arabidopsis. Here we report the identification of RRTF1 as an epigenetic target of RSI1. RRTF1 expression is higher in pathogen-free distal tissues of the rsi1 mutant. Experiments with loss-of-function and overexpression lines suggest RRTF1 is a negative regulator of basal defense against virulent and avirulent pathogens as well as SAR. Enhanced expression of RRTF1 in a wild-type (WT) background specifically impairs SAR without impacting local resistance. RSI1 is recruited at the RRTF1 locus in a SAR-inducible manner and contributes to H3K4me2 and H3K4me3 demethylation. Introduction of the rrtf1 mutation rescues the loss-of-SAR phenotype of rsi1 plants. However, these plants fail to retain infection memory beyond 7 days post-primary inoculation, whereas WT plants retain memory for at least 11 days. Our results demonstrate that RSI1 and RRTF1 form a functional module for retaining infection memory in Arabidopsis., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Here we report creation of a unique and a very valuable resource for Plant Scientific community worldwide. In this era of post-genomics and modelling of multi-cellular systems using an integrative systems biology approach, better understanding of protein localization at sub-cellular, cellular and tissue levels is likely to result in better understanding of their function and role in cell and tissue dynamics, protein-protein interactions and protein regulatory networks. We have raised 94 antibodies against key Arabidopsis root proteins, using either small peptides or recombinant proteins. The success rate with the peptide antibodies was very low. We show that affinity purification of antibodies massively improved the detection rate. Of 70 protein antibodies, 38 (55%) antibodies could detect a signal with high confidence and 22 of these antibodies are of immunocytochemistry grade. The targets include key proteins involved in hormone synthesis, transport and perception, membrane trafficking related proteins and several sub cellular marker proteins. These antibodies are available from the Nottingham Arabidopsis Stock Centre.

Drought is one of the major abiotic stresses which negatively affect plant growth and crop yield. Metallothionein (MTs) is a low molecular weight protein, mainly involved in metal homeostasis, while, its role in drought stress is still to be largely explored. The present study was aimed to investigate the role of MT gene against drought stress. The chickpea MT based on its up-regulation under drought stress was overexpressed in Arabidopsis thaliana to explore its role in mitigation of drought stress. The total transcript of MT gene was up to 30 fold higher in transgenic lines. Arabidopsis plants transformed with MT gene showed longer roots, better efficiency of survival and germination, larger siliques and higher biomass compared to WT. The physiological variables (A, WUE, G, E, qP and ETR) of WT plants were reduced during drought stress which recovered in transgenic Arabidopsis lines. The enzymatic and non-enzymatic antioxidant (APX, GPX, POD, GR, GRX, GST, CAT, MDHAR, ASc and GSH) levels were also enhanced in transgenic lines to provide tolerance. Simultaneously, drought responsive amino acids, i.e. proline and cysteine contents were higher in transgenic lines. Overall, the results suggest that MT gene is actively involved in the mitigation of drought stress and could be the choice for genetic engineering strategy to overcome drought stress., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Phosphate (P) is an essential macronutrient for plant growth. Roots employ adaptive mechanisms to forage for P in soil. Root hair elongation is particularly important since P is immobile. Here we report that auxin plays a critical role promoting root hair growth in Arabidopsis in response to low external P. Mutants disrupting auxin synthesis (taa1) and transport (aux1) attenuate the low P root hair response. Conversely, targeting AUX1 expression in lateral root cap and epidermal cells rescues this low P response in aux1. Hence auxin transport from the root apex to differentiation zone promotes auxin-dependent hair response to low P. Low external P results in induction of root hair expressed auxin-inducible transcription factors ARF19, RSL2, and RSL4. Mutants lacking these genes disrupt the low P root hair response. We conclude auxin synthesis, transport and response pathway components play critical roles regulating this low P root adaptive response.

Root hairs facilitate a plant's ability to acquire soil anchorage and nutrients. Root hair growth is regulated by the plant hormone auxin and dependent on localized synthesis, secretion, and modification of the root hair tip cell wall. However, the exact cell wall regulators in root hairs controlled by auxin have yet to be determined. In this study, we describe the characterization of ERULUS (ERU), an auxin-induced Arabidopsis receptor-like kinase, whose expression is directly regulated by ARF7 and ARF19 transcription factors. ERU belongs to the Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) subfamily of putative cell wall sensor proteins. Imaging of a fluorescent fusion protein revealed that ERU is localized to the apical root hair plasma membrane. ERU regulates cell wall composition in root hairs and modulates pectin dynamics through negative control of pectin methylesterase (PME) activity. Mutant eru (-/-) root hairs accumulate de-esterified homogalacturonan and exhibit aberrant pectin Ca 2+ -binding site oscillations and increased PME activity. Up to 80% of the eru root hair phenotype is rescued by pharmacological supplementation with a PME-inhibiting catechin extract. ERU transcription is altered in specific cell wall-related root hair mutants, suggesting that it is a target for feedback regulation. Loss of ERU alters the phosphorylation status of FERONIA and H + -ATPases 1/2, regulators of apoplastic pH. Furthermore, H + -ATPases 1/2 and ERU are differentially phosphorylated in response to auxin. We conclude that ERULUS is a key auxin-controlled regulator of cell wall composition and pectin dynamics during root hair tip growth., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Auxin represents a key signal in plants, regulating almost every aspect of their growth and development. Major breakthroughs have been made dissecting the molecular basis of auxin transport, perception, and response. In contrast, how plants control the metabolism and homeostasis of the major form of auxin in plants, indole-3-acetic acid (IAA), remains unclear. In this paper, we initially describe the function of the Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1 (AtDAO1). Transcriptional and translational reporter lines revealed that AtDAO1 encodes a highly root-expressed, cytoplasmically localized IAA oxidase. Stable isotope-labeled IAA feeding studies of loss and gain of function AtDAO1 lines showed that this oxidase represents the major regulator of auxin degradation to 2-oxoindole-3-acetic acid (oxIAA) in Arabidopsis Surprisingly, AtDAO1 loss and gain of function lines exhibited relatively subtle auxin-related phenotypes, such as altered root hair length. Metabolite profiling of mutant lines revealed that disrupting AtDAO1 regulation resulted in major changes in steady-state levels of oxIAA and IAA conjugates but not IAA. Hence, IAA conjugation and catabolism seem to regulate auxin levels in Arabidopsis in a highly redundant manner. We observed that transcripts of AtDOA1 IAA oxidase and GH3 IAA-conjugating enzymes are auxin-inducible, providing a molecular basis for their observed functional redundancy. We conclude that the AtDAO1 gene plays a key role regulating auxin homeostasis in Arabidopsis, acting in concert with GH3 genes, to maintain auxin concentration at optimal levels for plant growth and development., Competing Interests: The authors declare no conflict of interest.

Lateral root primordia (LRP) originate from pericycle stem cells located deep within parental root tissues. LRP emerge through overlying root tissues by inducing auxin-dependent cell separation and hydraulic changes in adjacent cells. The auxin-inducible auxin influx carrier LAX3 plays a key role concentrating this signal in cells overlying LRP. Delimiting LAX3 expression to two adjacent cell files overlying new LRP is crucial to ensure that auxin-regulated cell separation occurs solely along their shared walls. Multiscale modeling has predicted that this highly focused pattern of expression requires auxin to sequentially induce auxin efflux and influx carriers PIN3 and LAX3, respectively. Consistent with model predictions, we report that auxin-inducible LAX3 expression is regulated indirectly by AUXIN RESPONSE FACTOR 7 (ARF7). Yeast one-hybrid screens revealed that the LAX3 promoter is bound by the transcription factor LBD29, which is a direct target for regulation by ARF7. Disrupting auxin-inducible LBD29 expression or expressing an LBD29-SRDX transcriptional repressor phenocopied the lax3 mutant, resulting in delayed lateral root emergence. We conclude that sequential LBD29 and LAX3 induction by auxin is required to coordinate cell separation and organ emergence., (© 2016. Published by The Company of Biologists Ltd.)

Leaf shape in Arabidopsis is modulated by patterning events in the margin that utilize a PIN-based auxin exporter/CUC2 transcription factor system to define regions of promotion and retardation of growth, leading to morphogenesis. In addition to auxin exporters, leaves also express auxin importers, notably members of the AUX1/LAX family. In contrast to their established roles in embryogenesis, lateral root and leaf initiation, the function of these transporters in leaf development is poorly understood. We report that three of these genes (AUX1, LAX1 and LAX2) show specific and dynamic patterns of expression during early leaf development in Arabidopsis, and that loss of expression of all three genes is required for observation of a phenotype in which morphogenesis (serration) is decreased. We used these expression patterns and mutant phenotypes to develop a margin-patterning model that incorporates an AUX1/LAX1/LAX2 auxin import module that influences the extent of leaf serration. Testing of this model by margin-localized expression of axr3-1 (AXR17) provides further insight into the role of auxin in leaf morphogenesis., (© 2015 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The female gametophyte of flowering plants, called the embryo sac, develops from a haploid cell named the functional megaspore, which is specified after meiosis by the diploid sporophyte. In Arabidopsis, the functional megaspore undergoes three syncitial mitotic divisions followed by cellularization to form seven cells of four cell types including two female gametes. The plant hormone auxin is important for sporophytic developmental processes, and auxin levels are known to be regulated by biosynthesis and transport. Here, we investigated the role of auxin biosynthetic genes and auxin influx carriers in embryo sac development. We find that genes from the YUCCA/TAA pathway (YUC1, YUC2, YUC8, TAA1, TAR2) are expressed asymmetrically in the developing ovule and embryo sac from the two-nuclear syncitial stage until cellularization. Mutants for YUC1 and YUC2 exhibited defects in cell specification, whereas mutations in YUC8, as well as mutations in TAA1 and TAR2, caused defects in nuclear proliferation, vacuole formation and anisotropic growth of the embryo sac. Additionally, expression of the auxin influx carriers AUX1 and LAX1 were observed at the micropylar pole of the embryo sac and in the adjacent cells of the ovule, and the aux1 lax1 lax2 triple mutant shows multiple gametophyte defects. These results indicate that both localized auxin biosynthesis and auxin import, are required for mitotic divisions, cell expansion and patterning during embryo sac development.

The plant hormone auxin and its directional transport are known to play a crucial role in defining the embryonic axis and subsequent development of the body plan. Although the role of PIN auxin efflux transporters has been clearly assigned during embryonic shoot and root specification, the role of the auxin influx carriers AUX1 and LIKE-AUX1 (LAX) proteins is not well established. Here, we used chemical and genetic tools on Brassica napus microspore-derived embryos and Arabidopsis thaliana zygotic embryos, and demonstrate that AUX1, LAX1 and LAX2 are required for both shoot and root pole formation, in concert with PIN efflux carriers. Furthermore, we uncovered a positive-feedback loop between MONOPTEROS (ARF5)-dependent auxin signalling and auxin transport. This MONOPTEROS-dependent transcriptional regulation of auxin influx (AUX1, LAX1 and LAX2) and auxin efflux (PIN1 and PIN4) carriers by MONOPTEROS helps to maintain proper auxin transport to the root tip. These results indicate that auxin-dependent cell specification during embryo development requires balanced auxin transport involving both influx and efflux mechanisms, and that this transport is maintained by a positive transcriptional feedback on auxin signalling., (© 2015. Published by The Company of Biologists Ltd.)

The stem cell niche of the Arabidopsis (Arabidopsis thaliana) primary root apical meristem is composed of the quiescent (or organizing) center surrounded by stem (initial) cells for the different tissues. Initial cells generate a population of transit-amplifying cells that undergo a limited number of cell divisions before elongating and differentiating. It is unclear whether these divisions occur stochastically or in an orderly manner. Using the thymidine analog 5-ethynyl-2'-deoxyuridine to monitor DNA replication of cells of Arabidopsis root meristems, we identified a pattern of two, four, and eight neighboring cells with synchronized replication along the cortical, epidermal, and endodermal cell files, suggested to be daughters, granddaughters, and great-granddaughters of the direct progeny of each stem cell. Markers of mitosis and cytokinesis were not present in the region closest to the transition zone where the cells start to elongate, suggesting that great-granddaughter cells switch synchronously from the mitotic cell cycle to endoreduplication. Mutations in the stem cell niche-expressed ASH1-RELATED3 (ASHR3) gene, encoding a SET-domain protein conferring histone H3 lysine-36 methylation, disrupted this pattern of coordinated DNA replication and cell division and increased the cell division rate in the quiescent center. E2Fa/E2Fb transcription factors controlling the G1-to-S-phase transition regulate ASHR3 expression and bind to the ASHR3 promoter, substantiating a role for ASHR3 in cell division control. The reduced length of the root apical meristem and primary root of the mutant ashr3-1 indicate that synchronization of replication and cell divisions is required for normal root growth and development., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

Auxin is a key regulator of plant growth and development. Within the root tip, auxin distribution plays a crucial role specifying developmental zones and coordinating tropic responses. Determining how the organ-scale auxin pattern is regulated at the cellular scale is essential to understanding how these processes are controlled. In this study, we developed an auxin transport model based on actual root cell geometries and carrier subcellular localizations. We tested model predictions using the DII-VENUS auxin sensor in conjunction with state-of-the-art segmentation tools. Our study revealed that auxin efflux carriers alone cannot create the pattern of auxin distribution at the root tip and that AUX1/LAX influx carriers are also required. We observed that AUX1 in lateral root cap (LRC) and elongating epidermal cells greatly enhance auxin's shootward flux, with this flux being predominantly through the LRC, entering the epidermal cells only as they enter the elongation zone. We conclude that the nonpolar AUX1/LAX influx carriers control which tissues have high auxin levels, whereas the polar PIN carriers control the direction of auxin transport within these tissues.

Plants survive adverse conditions by modulating their growth in response to a changing environment. Gibberellins (GAs) play a key role in these adaptive responses by stimulating the degradation of growth-repressing DELLA proteins. GA binding to its receptor GID1 enables association of GID1 with DELLAs. This leads to the ubiquitin-mediated proteasomal degradation of DELLAs and consequently growth promotion. We report that DELLA-dependent growth control can be regulated independently of GA. We demonstrate that when a proportion of DELLAs is conjugated to the Small Ubiquitin-like Modifier (SUMO) protein, the extent of conjugation increases during stress. We identify a SUMO-interacting motif in GID1 and demonstrate that SUMO-conjugated DELLA binds to this motif in a GA-independent manner. The consequent sequestration of GID1 by SUMO-conjugated DELLAs leads to an accumulation of non-SUMOylated DELLAs, resulting in beneficial growth restraint during stress. We conclude that plants have developed a GA-independent mechanism to control growth., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: In the root apical meristem, which contains the stem cells that feed into root development, the phytohormones auxin and cytokinin play opposing roles, with auxin promoting cell division and cytokinin promoting cell differentiation. Cytokinin acts in the root tip in part by modulating auxin transport through regulation of the level of the PIN auxin efflux carriers. Auxin plays a key role in the specification of the quiescent center (QC), which is essential for maintaining the stem cell fate of the surrounding cells., Results: We demonstrate that cytokinin promotes cell division in the QC, which is generally mitotically inactive. Cytokinin downregulates the expression of several key regulatory genes in the root tip, including SCARECROW, WOX5, and the auxin influx carriers AUX1 and LAX2. The decrease in LAX2 expression in response to cytokinin requires ARR1 and ARR12, two type B ARRs that mediate the primary transcriptional response to cytokinin. ARR1 was found to bind directly to the LAX2 gene in vivo, which indicates that type B ARRs directly regulate genes that are repressed by cytokinin. Disruption of the LAX2 gene results in a phenotype similar to that observed in response to cytokinin, including increased division of the cells in the QC and decreased expression of WOX5 and the auxin response reporter DR5., Conclusions: Cytokinin acts to regulate auxin distribution in the root apical meristem by regulating both the PINs and LAX2. This redistribution of auxin, potentially coupled with other auxin-independent effects of cytokinin, regulates the mitotic activity in the QC., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The plant hormone indole-acetic acid (auxin) is essential for many aspects of plant development. Auxin-mediated growth regulation typically involves the establishment of an auxin concentration gradient mediated by polarly localized auxin transporters. The localization of auxin carriers and their amount at the plasma membrane are controlled by membrane trafficking processes such as secretion, endocytosis, and recycling. In contrast to endocytosis or recycling, how the secretory pathway mediates the localization of auxin carriers is not well understood. In this study we have used the differential cell elongation process during apical hook development to elucidate the mechanisms underlying the post-Golgi trafficking of auxin carriers in Arabidopsis. We show that differential cell elongation during apical hook development is defective in Arabidopsis mutant echidna (ech). ECH protein is required for the trans-Golgi network (TGN)-mediated trafficking of the auxin influx carrier AUX1 to the plasma membrane. In contrast, ech mutation only marginally perturbs the trafficking of the highly related auxin influx carrier LIKE-AUX1-3 or the auxin efflux carrier PIN-FORMED-3, both also involved in hook development. Electron tomography reveals that the trafficking defects in ech mutant are associated with the perturbation of secretory vesicle genesis from the TGN. Our results identify differential mechanisms for the post-Golgi trafficking of de novo-synthesized auxin carriers to plasma membrane from the TGN and reveal how trafficking of auxin influx carriers mediates the control of differential cell elongation in apical hook development.

The phytohormone auxin regulates virtually every aspect of plant development. To identify new genes involved in auxin activity, a genetic screen was performed for Arabidopsis (Arabidopsis thaliana) mutants with altered expression of the auxin-responsive reporter DR5rev:GFP. One of the mutants recovered in the screen, designated as weak auxin response3 (wxr3), exhibits much lower DR5rev:GFP expression when treated with the synthetic auxin 2,4-dichlorophenoxyacetic acid and displays severe defects in root development. The wxr3 mutant decreases polar auxin transport and results in a disruption of the asymmetric auxin distribution. The levels of the auxin transporters AUXIN1 and PIN-FORMED are dramatically reduced in the wxr3 root tip. Molecular analyses demonstrate that WXR3 is ROOT ULTRAVIOLET B-SENSITIVE1 (RUS1), a member of the conserved Domain of Unknown Function647 protein family found in diverse eukaryotic organisms. Our data suggest that RUS1/WXR3 plays an essential role in the regulation of polar auxin transport by maintaining the proper level of auxin transporters on the plasma membrane.

Auxin transport, which is mediated by specialized influx and efflux carriers, plays a major role in many aspects of plant growth and development. AUXIN1 (AUX1) has been demonstrated to encode a high-affinity auxin influx carrier. In Arabidopsis thaliana, AUX1 belongs to a small multigene family comprising four highly conserved genes (i.e., AUX1 and LIKE AUX1 [LAX] genes LAX1, LAX2, and LAX3). We report that all four members of this AUX/LAX family display auxin uptake functions. Despite the conservation of their biochemical function, AUX1, LAX1, and LAX3 have been described to regulate distinct auxin-dependent developmental processes. Here, we report that LAX2 regulates vascular patterning in cotyledons. We also describe how regulatory and coding sequences of AUX/LAX genes have undergone subfunctionalization based on their distinct patterns of spatial expression and the inability of LAX sequences to rescue aux1 mutant phenotypes, respectively. Despite their high sequence similarity at the protein level, transgenic studies reveal that LAX proteins are not correctly targeted in the AUX1 expression domain. Domain swapping studies suggest that the N-terminal half of AUX1 is essential for correct LAX localization. We conclude that Arabidopsis AUX/LAX genes encode a family of auxin influx transporters that perform distinct developmental functions and have evolved distinct regulatory mechanisms.

Gravity profoundly influences plant growth and development. Plants respond to changes in orientation by using gravitropic responses to modify their growth. Cholodny and Went hypothesized over 80 years ago that plants bend in response to a gravity stimulus by generating a lateral gradient of a growth regulator at an organ's apex, later found to be auxin. Auxin regulates root growth by targeting Aux/IAA repressor proteins for degradation. We used an Aux/IAA-based reporter, domain II (DII)-VENUS, in conjunction with a mathematical model to quantify auxin redistribution following a gravity stimulus. Our multidisciplinary approach revealed that auxin is rapidly redistributed to the lower side of the root within minutes of a 90° gravity stimulus. Unexpectedly, auxin asymmetry was rapidly lost as bending root tips reached an angle of 40° to the horizontal. We hypothesize roots use a "tipping point" mechanism that operates to reverse the asymmetric auxin flow at the midpoint of root bending. These mechanistic insights illustrate the scientific value of developing quantitative reporters such as DII-VENUS in conjunction with parameterized mathematical models to provide high-resolution kinetics of hormone redistribution.

Short-Root (SHR) is a well-characterized regulator of radial patterning and indeterminacy of the Arabidopsis (Arabidopsis thaliana) primary root. However, its role during the elaboration of root system architecture remains unclear. We report that the indeterminate wild-type Arabidopsis root system was transformed into a determinate root system in the shr mutant when growing in soil or agar. The root growth behavior of the shr mutant results from its primary root apical meristem failing to initiate cell division following germination. The inability of shr to reactivate mitotic activity in the root apical meristem is associated with the progressive reduction in the abundance of auxin efflux carriers, PIN-FORMED1 (PIN1), PIN2, PIN3, PIN4, and PIN7. The loss of primary root growth in shr is compensated by the activation of anchor root primordia, whose tissues are radially patterned like the wild type. However, SHR function is not restricted to the primary root but is also required for the initiation and patterning of lateral root primordia. In addition, SHR is necessary to maintain the indeterminate growth of lateral and anchor roots. We conclude that SHR regulates a wide array of Arabidopsis root-related developmental processes.

Background and Aims: The root meristem of the Arabidopsis thaliana mature embryo is a highly organized structure in which individual cell shape and size must be regulated in co-ordination with the surrounding cells. The objective of this study was to determine the role of the AUX1 LAX family of auxin import carriers during the establishment of the embryonic root cell pattern., Methods: The radicle apex of single and multiple aux1 lax mutant mature embryos was used to evaluate the effect of this gene family upon embryonic root organization and root cap size, cell number and cell size., Key Results: It was demonstrated here that mutations within the AUX1 LAX family are associated with changes in cell pattern establishment in the embryonic quiescent centre and columella. aux1 lax mutants have a larger radicle root cap than the wild type and this is associated with a significant increase in the root-cap cell number, average cell size, or both. Extreme disorganization of the radicle apex was observed among quadruple aux1 lax1 lax2 lax3 mutant embryos, but not in single aux1 null or in lax1, lax2 and lax3 single mutants, indicating redundancy within the AUX1 LAX family., Conclusions: It was determined that the AUX1 LAX family of auxin influx facilitators participates in the establishment of cell pattern within the apex of the embryonic root in a gene-redundant fashion. It was demonstrated that aux1 lax mutants are affected in cell proliferation and cell growth within the radicle tip. Thus AUX1 LAX auxin importers emerge as new players in morphogenetic processes involved in patterning during embryonic root formation.

Dark-grown dicotyledonous seedlings form a hook-like structure at the top of the hypocotyl, which is controlled by the hormones auxin and ethylene. Hook formation is dependent on an auxin signal gradient, whereas hook exaggeration is part of the triple response provoked by ethylene in dark-grown Arabidopsis seedlings. Several other hormones and light are also known to be involved in hook development, but the molecular mechanisms that lead to the initial installation of an auxin gradient are still poorly understood. In this study, we aimed to unravel the cross-talk between auxin and ethylene in the apical hook. Auxin measurements, the expression pattern of the auxin reporter DR5::GUS and the localization of auxin biosynthesis enzymes and influx carriers collectively indicate the necessity for auxin biosynthesis and efficient auxin translocation from the cotyledons and meristem into the hypocotyl in order to support proper hook development. Auxin accumulation in the meristem and cotyledons and in the hypocotyl is increased approximately 2-fold upon treatment with ethylene. In addition, a strong ethylene signal leads to enhanced auxin biosynthesis at the inner side of the hook. Finally, mutant analysis demonstrates that the auxin influx carrier LAX3 is indispensable for proper hook formation, whereas the auxin influx carrier AUX1 is involved in the hook exaggeration phenotype induced by ethylene.

Plant growth is driven by cell proliferation and elongation. The hormone gibberellin (GA) regulates Arabidopsis root growth by controlling cell elongation, but it is currently unknown whether GA also controls root cell proliferation. Here we show that GA biosynthetic mutants are unable to increase their cell production rate and meristem size after germination. GA signals the degradation of the DELLA growth repressor proteins GAI and RGA, promoting root cell production. Targeting the expression of gai (a non-GA-degradable mutant form of GAI) in the root meristem disrupts cell proliferation. Moreover, expressing gai in dividing endodermal cells was sufficient to block root meristem enlargement. We report a novel function for GA regulating cell proliferation where this signal acts by removing DELLA in a subset of, rather than all, meristem cells. We suggest that the GA-regulated rate of expansion of dividing endodermal cells dictates the equivalent rate in other root tissues. Cells must double in size prior to dividing but cannot do so independently, because they are physically restrained by adjacent tissues with which they share cell walls. Our study highlights the importance of probing regulatory mechanisms linking molecular- and cellular-scale processes with tissue and organ growth responses.

Lateral root formation is a major determinant of root systems architecture. The degree of root branching impacts the efficiency of water uptake, acquisition of nutrients and anchorage by plants. Understanding the regulation of lateral root development is therefore of vital agronomic importance. The molecular and cellular basis of lateral root formation has been most extensively studied in the plant model Arabidopsis thaliana (Arabidopsis). Significant progress has recently been made in identifying many new Arabidopsis genes that regulate lateral root initiation, patterning and emergence processes. We review how these studies have revealed that the plant hormone auxin represents a common signal that integrates these distinct yet interconnected developmental processes.

The plant hormone auxin controls root epidermal cell development in a concentration-dependent manner. Root hairs are produced on a subset of epidermal cells as they increase in distance from the root tip. Auxin is required for their initiation and continued growth, but little is known about its distribution in this region of the root. Contrary to the expectation that hair cells might require active auxin influx to ensure auxin supply, we did not detect the auxin-influx transporter AUX1 in root-hair cells. A high level of AUX1 expression was detected in adjacent non-hair cell files. Non-hair cells were necessary to achieve wild-type root-hair length, although an auxin response was not required in these cells. Three-dimensional modelling of auxin flow in the root tip suggests that AUX1-dependent transport through non-hair cells maintains an auxin supply to developing hair cells as they increase in distance from the root tip, and sustains root-hair outgrowth. Experimental data support the hypothesis that instead of moving uniformly though the epidermal cell layer, auxin is mainly transported through canals that extend longitudinally into the tissue.

The cellular import of the hormone auxin is a fundamental requirement for the generation of auxin gradients that control a multitude of plant developmental processes. The AUX/LAX family of auxin importers, exemplified by AUX1 from Arabidopsis (Arabidopsis thaliana), has been shown to mediate auxin import when expressed heterologously. The quantitative nature of the interaction between AUX1 and its transport substrate indole-3-acetic acid (IAA) is incompletely understood, and we sought to address this in the present investigation. We expressed AUX1 to high levels in a baculovirus expression system and prepared membrane fragments from baculovirus-infected insect cells. These membranes proved suitable for determination of the binding of IAA to AUX1 and enabled us to determine a K(d) of 2.6 mum, comparable with estimates for the K(m) for IAA transport. The efficacy of a number of auxin analogues and auxin transport inhibitors to displace IAA binding from AUX1 has also been determined and can be rationalized in terms of their physiological effects. Determination of the parameters describing the initial interaction between a plant transporter and its hormone ligand provides novel quantitative data for modeling auxin fluxes.

Gibberellins (GAs) are key regulators of plant growth and development. They promote growth by targeting the degradation of DELLA repressor proteins; however, their site of action at the cellular, tissue or organ levels remains unknown. To map the site of GA action in regulating root growth, we expressed gai, a non-degradable, mutant DELLA protein, in selected root tissues. Root growth was retarded specifically when gai was expressed in endodermal cells. Our results demonstrate that the endodermis represents the primary GA-responsive tissue regulating organ growth and that endodermal cell expansion is rate-limiting for elongation of other tissues and therefore of the root as a whole.

One of the most striking features of plant architecture is the regular arrangement of leaves and flowers around the stem, known as phyllotaxis. Peaks in concentration of the plant hormone auxin, generated by the polar localization of the PIN1 auxin efflux carrier, provide the instructive signal for primordium initiation. This mechanism generates the spacing between neighboring primordia, which results in regular phyllotaxis. Studies of the role of auxin transport in phyllotactic patterning have focused on PIN1-mediated efflux. Recent computer simulations indicate an additional role for transporter-mediated auxin uptake. Mutations in the AUX1 auxin influx carrier have not, however, been reported to cause an aerial phenotype. Here, we study the role of AUX1 and its paralogs LAX1, LAX2, and LAX3. Analysis of the quadruple mutant reveals irregular divergence angles between successive primordia. A highly unusual aspect of the phenotype is the occurrence of clusters of primordia, in violation of classical theory. At the molecular level, the sharp peaks in auxin levels and coordinated PIN polarization are reduced or lost. In addition, the increased penetrance of the phenotype under short-day conditions suggests that the AUX LAX transporters act to buffer the PIN-mediated patterning mechanism against environmental or developmental influences.

In Arabidopsis thaliana, lateral roots are formed from root pericycle cells adjacent to the xylem poles. Lateral root development is regulated antagonistically by the plant hormones auxin and cytokinin. While a great deal is known about how auxin promotes lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to disrupt lateral root initiation and the regular pattern of divisions that characterizes lateral root development in Arabidopsis. To identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.

Ethylene represents an important regulatory signal for root development. Genetic studies in Arabidopsis thaliana have demonstrated that ethylene inhibition of root growth involves another hormone signal, auxin. This study investigated why auxin was required by ethylene to regulate root growth. We initially observed that ethylene positively controls auxin biosynthesis in the root apex. We subsequently demonstrated that ethylene-regulated root growth is dependent on (1) the transport of auxin from the root apex via the lateral root cap and (2) auxin responses occurring in multiple elongation zone tissues. Detailed growth studies revealed that the ability of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid to inhibit root cell elongation was significantly enhanced in the presence of auxin. We conclude that by upregulating auxin biosynthesis, ethylene facilitates its ability to inhibit root cell expansion.

The directional flow of the plant hormone auxin mediates multiple developmental processes, including patterning and tropisms. Apical and basal plasma membrane localization of AUXIN-RESISTANT1 (AUX1) and PIN-FORMED1 (PIN1) auxin transport components underpins the directionality of intercellular auxin flow in Arabidopsis thaliana roots. Here, we examined the mechanism of polar trafficking of AUX1. Real-time live cell analysis along with subcellular markers revealed that AUX1 resides at the apical plasma membrane of protophloem cells and at highly dynamic subpopulations of Golgi apparatus and endosomes in all cell types. Plasma membrane and intracellular pools of AUX1 are interconnected by actin-dependent constitutive trafficking, which is not sensitive to the vesicle trafficking inhibitor brefeldin A. AUX1 subcellular dynamics are not influenced by the auxin influx inhibitor NOA but are blocked by the auxin efflux inhibitors TIBA and PBA. Furthermore, auxin transport inhibitors and interference with the sterol composition of membranes disrupt polar AUX1 distribution at the plasma membrane. Compared with PIN1 trafficking, AUX1 dynamics display different sensitivities to trafficking inhibitors and are independent of the endosomal trafficking regulator ARF GEF GNOM. Hence, AUX1 uses a novel trafficking pathway in plants that is distinct from PIN trafficking, providing an additional mechanism for the fine regulation of auxin transport.

We have investigated the subcellular localization, the domain topology, and the amino acid residues that are critical for the function of the presumptive Arabidopsis thaliana auxin influx carrier AUX1. Biochemical fractionation experiments and confocal studies using an N-terminal yellow fluorescent protein (YFP) fusion observed that AUX1 colocalized with plasma membrane (PM) markers. Because of its PM localization, we were able to take advantage of the steep pH gradient that exists across the plant cell PM to investigate AUX1 topology using YFP as a pH-sensitive probe. The YFP-coding sequence was inserted in selected AUX1 hydrophilic loops to orient surface domains on either apoplastic or cytoplasmic faces of the PM based on the absence or presence of YFP fluorescence, respectively. We were able to demonstrate in conjunction with helix prediction programs that AUX1 represents a polytopic membrane protein composed of 11 transmembrane spanning domains. In parallel, a large aux1 allelic series containing null, partial-loss-of-function, and conditional mutations was characterized to identify the functionally important domains and amino acid residues within the AUX1 polypeptide. Whereas almost all partial-loss-of-function and null alleles cluster in the core permease region, the sole conditional allele aux1-7 modifies the function of the external C-terminal domain.

Our aim was to generate and prove the concept of "smart" plants to monitor plant phosphorus (P) status in Arabidopsis. Smart plants can be genetically engineered by transformation with a construct containing the promoter of a gene up-regulated specifically by P starvation in an accessible tissue upstream of a marker gene such as beta-glucuronidase (GUS). First, using microarrays, we identified genes whose expression changed more than 2.5-fold in shoots of plants growing hydroponically when P, but not N or K, was withheld from the nutrient solution. The transient changes in gene expression occurring immediately (4 h) after P withdrawal were highly variable, and many nonspecific, shock-induced genes were up-regulated during this period. However, two common putative cis-regulatory elements (a PHO-like element and a TATA box-like element) were present significantly more often in the promoters of genes whose expression increased 4 h after the withdrawal of P compared with their general occurrence in the promoters of all genes represented on the microarray. Surprisingly, the expression of only four genes differed between shoots of P-starved and -replete plants 28 h after P was withdrawn. This lull in differential gene expression preceded the differential expression of a new group of 61 genes 100 h after withdrawing P. A literature survey indicated that the expression of many of these "late" genes responded specifically to P starvation. Shoots had reduced P after 100 h, but growth was unaffected. The expression of SQD1, a gene involved in the synthesis of sulfolipids, responded specifically to P starvation and was increased 100 h after withdrawing P. Leaves of Arabidopsis bearing a SQD1::GUS construct showed increased GUS activity after P withdrawal, which was detectable before P starvation limited growth. Hence, smart plants can monitor plant P status. Transferring this technology to crops would allow precision management of P fertilization, thereby maintaining yields while reducing costs, conserving natural resources, and preventing pollution.

Coordination of cell and tissue polarity commonly involves directional signaling. In the Arabidopsis root epidermis, cell polarity is revealed by basal, root tip-oriented, hair outgrowth from hair-forming cells (trichoblasts). The plant hormone auxin displays polar movements and accumulates at maximum concentration in the root tip. The application of polar auxin transport inhibitors evokes changes in trichoblast polarity only at high concentrations and after long-term application. Thus, it remains open whether components of the auxin transport machinery mediate establishment of trichoblast polarity. Here we report that the presumptive auxin influx carrier AUX1 contributes to apical-basal hair cell polarity. AUX1 function is required for polarity changes induced by exogenous application of the auxin 2,4-D, a preferential influx carrier substrate. Similar to aux1 mutants, the vesicle trafficking inhibitor brefeldin A (BFA) interferes with polar hair initiation, and AUX1 function is required for BFA-mediated polarity changes. Consistently, BFA inhibits membrane trafficking of AUX1, trichoblast hyperpolarization induced by 2,4-D, and alters the distal auxin maximum. Our results identify AUX1 as one component of a novel BFA-sensitive auxin transport pathway polarizing cells toward a hormone maximum.

During leaf abscission in oilseed rape (Brassica napus), cell wall degradation is brought about by the action of several hydrolytic enzymes. One of these is thought to be polygalacturonase (PG). Degenerate primers were used to isolate a PG cDNA fragment by reverse transcriptase-polymerase chain reaction from RNA extracted from ethylene-promoted leaf abscission zones (AZs), and in turn a full-length clone (CAW471) from an oilseed rape AZ cDNA library. The highest homology of this cDNA (82%) was to an Arabidopsis sequence that was predicted to encode a PG protein. Analysis of expression revealed that CAW471 mRNA accumulated in the AZ of leaves and reached a peak 24 h after ethylene treatment. Ethylene-promoted leaf abscission in oilseed rape was not apparent until 42 h after exposure to the gas, reaching 50% at 48 h and 100% by 56 h. In floral organ abscission, expression of CAW471 correlated with cell separation. Genomic libraries from oilseed rape and Arabidopsis were screened with CAW471 and the respective genomic clones PGAZBRAN and PGAZAT isolated. Characterization of these PG genes revealed that they had substantial homology within both the coding regions and in the 5'-upstream sequences. Fusion of a 1,476-bp 5'-upstream sequence of PGAZAT to beta-glucuronidase or green fluorescent protein and transformation of Arabidopsis revealed that this fragment was sufficient to drive expression of these reporter genes in the AZs at the base of the anther filaments, petals, and sepals.

SFR2, a member of the S gene family of receptor kinases, has been shown to be rapidly induced by wounding and bacterial infection suggesting that this gene may play a role in the defence response in Brassica. In this study we have compared the response of SFR2 to that of two other members of the SFR gene family in Brassica (SFR1 and SFR3) and to the closely-related ARK genes of Arabidopsis. Different patterns of mRNA accumulation were observed for different members of these families. SFR1 transcripts only accumulated in response to bacterial infection and their abundance was not significantly affected by wounding. Neither treatment induced accumulation of SFR3 transcripts. ARK1 and ARK3 resembled SFR2 in that their mRNAs accumulated in response to both wounding and bacterial infection. Both SFR1 and SFR2 mRNAs accumulated in response to exogenously applied salicylic acid (SA) and SA was shown to be required for induction of expression from the SFR2 promoter in Arabidopsis. However, the timing of the increase in endogenous SA levels following bacterial infiltration in Brassica indicates that the accumulation of SFR mRNA in the first few hours after infiltration does not occur in response to an increase in SA levels. We discuss the possibility that induction of SFR gene expression by SA may contribute to potentialization of the defence response. Taken together with previous studies that indicate a possible role during development, the data presented here suggest that the SFR and ARK gene families may have overlapping roles in both defence and during development.