Altered expression levels of long non-coding natural antisense transcripts overlapping the UGT73C6 gene affect rosette size in Arabidopsis thaliana.

Natural antisense long non-coding RNAs (lncNATs) are involved in the regulation of gene expression in plants, modulating different relevant developmental processes and responses to various stimuli. We have identified and characterized two lncNATs (NAT1UGT73C6 and NAT2UGT73C6 , collectively NATsUGT73C6 ) from Arabidopsis thaliana that are transcribed from a gene fully overlapping UGT73C6, a member of the UGT73C subfamily of genes encoding UDP-glycosyltransferases (UGTs). Expression of both NATsUGT73C6 is developmentally controlled and occurs independently of the transcription of UGT73C6 in cis. Downregulation of NATsUGT73C6 levels through artificial microRNAs results in a reduction of the rosette area, while constitutive overexpression of NAT1UGT73C6 or NAT2UGT73C6 leads to the opposite phenotype, an increase in rosette size. This activity of NATsUGT73C6 relies on its RNA sequence and, although modulation of UGT73C6 in cis cannot be excluded, the observed phenotypes are not a consequence of the regulation of UGT73C6 in trans. The NATsUGT73C6 levels were shown to affect cell proliferation and thus individual leaf size. Consistent with this concept, our data suggest that the NATsUGT73C6 influence the expression levels of key transcription factors involved in regulating leaf growth by modulating cell proliferation. These findings thus reveal an additional regulatory layer on the process of leaf growth. In this work, we characterized at the molecular level two long non-coding RNAs (NATsUGT73C6 ) that are transcribed in the opposite direction to UGT73C6, a gene encoding a glucosyltransferase involved in brassinosteroid homeostasis in A. thaliana. Our results indicate that NATsUGT73C6 expression influences leaf growth by acting in trans and by modulating the levels of transcription factors that are involved in the regulation of cell proliferation.

Endoplasmic reticulum calnexins participate in the primary root growth response to phosphate deficiency.

Accumulation of incompletely folded proteins in the endoplasmic reticulum (ER) leads to ER stress, activates ER protein degradation pathways, and upregulates genes involved in protein folding. This process is known as the unfolded protein response (UPR). The role of ER protein folding in plant responses to nutrient deficiencies is unclear. We analyzed Arabidopsis (Arabidopsis thaliana) mutants affected in ER protein quality control and established that both CALNEXIN (CNX) genes function in the primary root response to phosphate (Pi) deficiency. CNX1 and CNX2 are homologous ER lectins promoting protein folding of N-glycosylated proteins via the recognition of the GlcMan9GlcNAc2 glycan. Growth of cnx1-1 and cnx2-2 single mutants was similar to that of the wild type under high and low Pi conditions, but the cnx1-1 cnx2-2 double mutant showed decreased primary root growth under low Pi conditions due to reduced meristematic cell division. This phenotype was specific to Pi deficiency; the double mutant responded normally to osmotic and salt stress. Expression of CNX2 mutated in amino acids involved in binding the GlcMan9GlcNAc2 glycan failed to complement the cnx1-1 cnx2-2 mutant. The root growth phenotype was Fe-dependent and was associated with root apoplastic Fe accumulation. Two genes involved in Fe-dependent inhibition of primary root growth under Pi deficiency, the ferroxidase LOW PHOSPHATE 1 (LPR1) and P5-type ATPase PLEIOTROPIC DRUG RESISTANCE 2 (PDR2) were epistatic to CNX1/CNX2. Overexpressing PDR2 failed to complement the cnx1-1 cnx2-2 root phenotype. The cnx1-1 cnx2-2 mutant showed no evidence of UPR activation, indicating a limited effect on ER protein folding. CNX might process a set of N-glycosylated proteins specifically involved in the response to Pi deficiency.

The stem cell niche transcription factor ETHYLENE RESPONSE FACTOR 115 participates in aluminum-induced terminal differentiation in Arabidopsis roots.

Aluminum-dependent stoppage of root growth requires the DNA damage response (DDR) pathway including the p53-like transcription factor SUPPRESSOR OF GAMMA RADIATION 1 (SOG1), which promotes terminal differentiation of the root tip in response to Al dependent cell death. Transcriptomic analyses identified Al-induced SOG1-regulated targets as candidate mediators of this growth arrest. Analysis of these factors either as loss-of-function mutants or by overexpression in the als3-1 background shows ERF115, which is a key transcription factor that in other scenarios is rate-limiting for damaged stem cell replenishment, instead participates in transition from an actively growing root to one that has terminally differentiated in response to Al toxicity. This is supported by a loss-of-function erf115 mutant raising the threshold of Al required to promote terminal differentiation of Al hypersensitive als3-1. Consistent with its key role in stoppage of root growth, a putative ERF115 barley ortholog is also upregulated following Al exposure, suggesting a conserved role for this ATR-dependent pathway in Al response. In contrast to other DNA damage agents, these results show that ERF115 and likely related family members are important determinants of terminal differentiation of the root tip following Al exposure and central outputs of the SOG1-mediated pathway in Al response.

Root responses to aluminium and iron stresses require the SIZ1 SUMO ligase to modulate the STOP1 transcription factor.

STOP1, an Arabidopsis transcription factor favouring root growth tolerance against Al toxicity, acts in the response to iron under low Pi (-Pi). Previous studies have shown that Al and Fe regulate the stability and accumulation of STOP1 in roots, and that the STOP1 protein is sumoylated by an unknown E3 ligase. Here, using a forward genetics suppressor screen, we identified the E3 SUMO (small ubiquitin-like modifier) ligase SIZ1 as a modulator of STOP1 signalling. Mutations in SIZ1 increase the expression of ALMT1 (a direct target of STOP1) and root growth responses to Al and Fe stress in a STOP1-dependent manner. Moreover, loss-of-function mutations in SIZ1 enhance the abundance of STOP1 in the root tip. However, no sumoylated STOP1 protein was detected by Western blot analysis in our sumoylation assay in Escherichia coli, suggesting the presence of a more sophisticated mechanism. We conclude that the sumo ligase SIZ1 negatively regulates STOP1 signalling, at least in part by modulating STOP1 protein in the root tip. Our results will allow a better understanding of this signalling pathway.

Modulation of Phosphate Deficiency-Induced Metabolic Changes by Iron Availability in Arabidopsis thaliana.

Concurrent suboptimal supply of several nutrients requires the coordination of nutrient-specific transcriptional, phenotypic, and metabolic changes in plants in order to optimize growth and development in most agricultural and natural ecosystems. Phosphate (Pi) and iron (Fe) deficiency induce overlapping but mostly opposing transcriptional and root growth responses in Arabidopsis thaliana. On the metabolite level, Pi deficiency negatively modulates Fe deficiency-induced coumarin accumulation, which is controlled by Fe as well as Pi deficiency response regulators. Here, we report the impact of Fe availability on seedling growth under Pi limiting conditions and on Pi deficiency-induced accumulation of amino acids and organic acids, which play important roles in Pi use efficiency. Fe deficiency in Pi replete conditions hardly changed growth and metabolite profiles in roots and shoots of Arabidopsis thaliana, but partially rescued growth under conditions of Pi starvation and severely modulated Pi deficiency-induced metabolic adjustments. Analysis of T-DNA insertion lines revealed the concerted coordination of metabolic profiles by regulators of Fe (FIT, bHLH104, BRUTUS, PYE) as well as of Pi (SPX1, PHR1, PHL1, bHLH32) starvation responses. The results show the interdependency of Pi and Fe availability and the interplay between Pi and Fe starvation signaling on the generation of plant metabolite profiles.

The Local Phosphate Deficiency Response Activates Endoplasmic Reticulum Stress-Dependent Autophagy.

Inorganic phosphate (Pi) is often a limiting plant nutrient. In members of the Brassicaceae family, such as Arabidopsis (Arabidopsis thaliana), Pi deprivation reshapes root system architecture to favor topsoil foraging. It does so by inhibiting primary root extension and stimulating lateral root formation. Root growth inhibition from phosphate (Pi) deficiency is triggered by iron-stimulated, apoplastic reactive oxygen species generation and cell wall modifications, which impair cell-to-cell communication and meristem maintenance. These processes require LOW PHOSPHATE RESPONSE1 (LPR1), a cell wall-targeted ferroxidase, and PHOSPHATE DEFICIENCY RESPONSE2 (PDR2), the single endoplasmic reticulum (ER)-resident P5-type ATPase (AtP5A), which is thought to control LPR1 secretion or activity. Autophagy is a conserved process involving the vacuolar degradation of cellular components. While the function of autophagy is well established under nutrient starvation (C, N, or S), it remains to be explored under Pi deprivation. Because AtP5A/PDR2 likely functions in the ER stress response, we analyzed the effect of Pi limitation on autophagy. Our comparative study of mutants defective in the local Pi deficiency response, ER stress response, and autophagy demonstrated that ER stress-dependent autophagy is rapidly activated as part of the developmental root response to Pi limitation and requires the genetic PDR2-LPR1 module. We conclude that Pi-dependent activation of autophagy in the root apex is a consequence of local Pi sensing and the associated ER stress response, rather than a means for systemic recycling of the macronutrient.

Microtubule-associated protein IQ67 DOMAIN5 regulates morphogenesis of leaf pavement cells in Arabidopsis thaliana.

Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation, and cell morphology. Their organization and dynamics are co-ordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization, and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced anisotropic expansion. Lastly, we demonstrate IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays and provide first evidence for important roles for calcium in regulation of PC morphogenesis. Our work identifies IQD5 as a novel player in PC shape regulation and, for the first time, links calcium signaling to developmental processes that regulate anisotropic growth in PCs.

The IQD Family of Calmodulin-Binding Proteins Links Calcium Signaling to Microtubules, Membrane Subdomains, and the Nucleus.

Calcium (Ca2+) signaling and dynamic reorganization of the cytoskeleton are essential processes for the coordination and control of plant cell shape and cell growth. Calmodulin (CaM) and closely related calmodulin-like (CML) polypeptides are principal sensors of Ca2+ signals. CaM/CMLs decode and relay information encrypted by the second messenger via differential interactions with a wide spectrum of targets to modulate their diverse biochemical activities. The plant-specific IQ67 DOMAIN (IQD) family emerged as possibly the largest class of CaM-interacting proteins with undefined molecular functions and biological roles. Here, we show that the 33 members of the IQD family in Arabidopsis (Arabidopsis thaliana) differentially localize, using green fluorescent protein (GFP)-tagged proteins, to multiple and distinct subcellular sites, including microtubule (MT) arrays, plasma membrane subdomains, and nuclear compartments. Intriguingly, the various IQD-specific localization patterns coincide with the subcellular patterns of IQD-dependent recruitment of CaM, suggesting that the diverse IQD members sequester Ca2+-CaM signaling modules to specific subcellular sites for precise regulation of Ca2+-dependent processes. Because MT localization is a hallmark of most IQD family members, we quantitatively analyzed GFP-labeled MT arrays in Nicotiana benthamiana cells transiently expressing GFP-IQD fusions and observed IQD-specific MT patterns, which point to a role of IQDs in MT organization and dynamics. Indeed, stable overexpression of select IQD proteins in Arabidopsis altered cellular MT orientation, cell shape, and organ morphology. Because IQDs share biochemical properties with scaffold proteins, we propose that IQD families provide an assortment of platform proteins for integrating CaM-dependent Ca2+ signaling at multiple cellular sites to regulate cell function, shape, and growth.

Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response.

The plant hormone auxin activates primary response genes by facilitating proteolytic removal of auxin/indole-3-acetic acid (AUX/IAA)-inducible repressors, which directly bind to transcriptional auxin response factors (ARF). Most AUX/IAA and ARF proteins share highly conserved C-termini mediating homotypic and heterotypic interactions within and between both protein families. The high-resolution NMR structure of C-terminal domains III and IV of the AUX/IAA protein PsIAA4 from pea (Pisum sativum) revealed a globular ubiquitin-like ß-grasp fold with homologies to the Phox and Bem1p (PB1) domain. The PB1 domain of wild-type PsIAA4 features two distinct surface patches of oppositely charged amino acid residues, mediating front-to-back multimerization via electrostatic interactions. Mutations of conserved basic or acidic residues on either face suppressed PsIAA4 PB1 homo-oligomerization in vitro and confirmed directional interaction of full-length PsIAA4 in vivo (yeast two-hybrid system). Mixing of oppositely mutated PsIAA4 PB1 monomers enabled NMR mapping of the negatively charged interface of the reconstituted PsIAA4 PB1 homodimer variant, whose stoichiometry (1:1) and equilibrium binding constant (KD ∼ 6.4 µM) were determined by isothermal titration calorimetry. In silico protein-protein docking studies based on NMR and yeast interaction data derived a model of the PsIAA4 PB1 homodimer, which is comparable with other PB1 domain dimers, but indicated considerable differences between the homodimeric interfaces of AUX/IAA and ARF PB1 domains. Our study provides an impetus for elucidating the molecular determinants that confer specificity to complex protein-protein interaction circuits between members of the two central families of transcription factors important to the regulation of auxin-responsive gene expression.

Comparative expression profiling reveals a role of the root apoplast in local phosphate response.

BACKGROUND: Plant adaptation to limited phosphate availability comprises a wide range of responses to conserve and remobilize internal phosphate sources and to enhance phosphate acquisition. Vigorous restructuring of root system architecture provides a developmental strategy for topsoil exploration and phosphate scavenging. Changes in external phosphate availability are locally sensed at root tips and adjust root growth by modulating cell expansion and cell division. The functionally interacting Arabidopsis genes, LOW PHOSPHATE RESPONSE 1 and 2 (LPR1/LPR2) and PHOSPHATE DEFICIENCY RESPONSE 2 (PDR2), are key components of root phosphate sensing. We recently demonstrated that the LOW PHOSPHATE RESPONSE 1 - PHOSPHATE DEFICIENCY RESPONSE 2 (LPR1-PDR2) module mediates apoplastic deposition of ferric iron (Fe(3+)) in the growing root tip during phosphate limitation. Iron deposition coincides with sites of reactive oxygen species generation and triggers cell wall thickening and callose accumulation, which interfere with cell-to-cell communication and inhibit root growth. RESULTS: We took advantage of the opposite phosphate-conditional root phenotype of the phosphate deficiency response 2 mutant (hypersensitive) and low phosphate response 1 and 2 double mutant (insensitive) to investigate the phosphate dependent regulation of gene and protein expression in roots using genome-wide transcriptome and proteome analysis. We observed an overrepresentation of genes and proteins that are involved in the regulation of iron homeostasis, cell wall remodeling and reactive oxygen species formation, and we highlight a number of candidate genes with a potential function in root adaptation to limited phosphate availability. Our experiments reveal that FERRIC REDUCTASE DEFECTIVE 3 mediated, apoplastic iron redistribution, but not intracellular iron uptake and iron storage, triggers phosphate-dependent root growth modulation. We further highlight expressional changes of several cell wall-modifying enzymes and provide evidence for adjustment of the pectin network at sites of iron accumulation in the root. CONCLUSION: Our study reveals new aspects of the elaborate interplay between phosphate starvation responses and changes in iron homeostasis. The results emphasize the importance of apoplastic iron redistribution to mediate phosphate-dependent root growth adjustment and suggest an important role for citrate in phosphate-dependent apoplastic iron transport. We further demonstrate that root growth modulation correlates with an altered expression of cell wall modifying enzymes and changes in the pectin network of the phosphate-deprived root tip, supporting the hypothesis that pectins are involved in iron binding and/or phosphate mobilization.

Non-targeted profiling of semi-polar metabolites in Arabidopsis root exudates uncovers a role for coumarin secretion and lignification during the local response to phosphate limitation.

Plants have evolved two major strategies to cope with phosphate (Pi) limitation. The systemic response, mainly comprising increased Pi uptake and metabolic adjustments for more efficient Pi use, and the local response, enabling plants to explore Pi-rich soil patches by reorganization of the root system architecture. Unlike previous reports, this study focused on root exudation controlled by the local response to Pi deficiency. To approach this, a hydroponic system separating the local and systemic responses was developed. Arabidopsis thaliana genotypes exhibiting distinct sensitivities to Pi deficiency could be clearly distinguished by their root exudate composition as determined by non-targeted reversed-phase ultraperformance liquid chromatography electrospray ionization quadrupole-time-of-flight mass spectrometry metabolite profiling. Compared with wild-type plants or insensitive low phosphate root 1 and 2 (lpr1 lpr2) double mutant plants, the hypersensitive phosphate deficiency response 2 (pdr2) mutant exhibited a reduced number of differential features in root exudates after Pi starvation, suggesting the involvement of PDR2-encoded P5-type ATPase in root exudation. Identification and analysis of coumarins revealed common and antagonistic regulatory pathways between Pi and Fe deficiency-induced coumarin secretion. The accumulation of oligolignols in root exudates after Pi deficiency was inversely correlated with Pi starvation-induced lignification at the root tips. The strongest oligolignol accumulation in root exudates was observed for the insensitive lpr1 lpr2 double mutant, which was accompanied by the absence of Pi deficiency-induced lignin deposition, suggesting a role of LPR ferroxidases in lignin polymerization during Pi starvation.

Comparative analysis of Arabidopsis UGT74 glucosyltransferases reveals a special role of UGT74C1 in glucosinolate biosynthesis.

The study of glucosinolates and their regulation has provided a powerful framework for the exploration of fundamental questions about the function, evolution, and ecological significance of plant natural products, but uncertainties about their metabolism remain. Previous work has identified one thiohydroximate S-glucosyltransferase, UGT74B1, with an important role in the core pathway, but also made clear that this enzyme functions redundantly and cannot be the sole UDP-glucose dependent glucosyltransferase (UGT) in glucosinolate synthesis. Here, we present the results of a nearly comprehensive in vitro activity screen of recombinant Arabidopsis Family 1 UGTs, which implicate other members of the UGT74 clade as candidate glucosinolate biosynthetic enzymes. Systematic genetic analysis of this clade indicates that UGT74C1 plays a special role in the synthesis of aliphatic glucosinolates, a conclusion strongly supported by phylogenetic and gene expression analyses. Finally, the ability of UGT74C1 to complement phenotypes and chemotypes of the ugt74b1-2 knockout mutant and to express thiohydroximate UGT activity in planta provides conclusive evidence for UGT74C1 being an accessory enzyme in glucosinolate biosynthesis with a potential function during plant adaptation to environmental challenge.

Arabidopsis calmodulin-binding protein IQ67-domain 1 localizes to microtubules and interacts with kinesin light chain-related protein-1.

Calcium (Ca(2+)) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (K(d) ∼ 0.6 µm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca(2+)/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.

Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays.

BACKGROUND: Agrobacterium tumefaciens-based transient assays have become a common tool for answering questions related to protein localization and gene expression in a cellular context. The use of these assays assumes that the transiently transformed cells are observed under relatively authentic physiological conditions and maintain 'normal' sub-cellular behaviour. Although this premise is widely accepted, the question of whether cellular organization and organelle morphology is altered in Agrobacterium-infiltrated cells has not been examined in detail. The first indications of an altered sub-cellular environment came from our observation that a common laboratory strain, GV3101(pMP90), caused a drastic increase in stromule frequency. Stromules, or 'stroma-filled-tubules' emanate from the surface of plastids and are sensitive to a variety of biotic and abiotic stresses. Starting from this observation, the goal of our experiments was to further characterize the changes to the cell resulting from short-term bacterial infestation, and to identify the factor responsible for eliciting these changes. RESULTS: Using a protocol typical of transient assays we evaluated the impact of GV3101(pMP90) infiltration on chloroplast behaviour and morphology in Nicotiana benthamiana. Our experiments confirmed that GV3101(pMP90) consistently induces stromules and alters plastid position relative to the nucleus. These effects were found to be the result of strain-dependant secretion of cytokinin and its accumulation in the plant tissue. Bacterial production of the hormone was found to be dependant on the presence of a trans-zeatin synthase gene (tzs) located on the Ti plasmid of GV3101(pMP90). Bacteria-derived cytokinins were also correlated with changes to both soluble sugar level and starch accumulation. CONCLUSION: Although we have chosen to focus on how transient Agrobacterium infestation alters plastid based parameters, these changes to the morphology and position of a single organelle, combined with the measured increases in sugar and starch content, suggest global changes to cell physiology. This indicates that cells visualized during transient assays may not be as 'normal' as was previously assumed. Our results suggest that the impact of the bacteria can be minimized by choosing Agrobacterium strains devoid of the tzs gene, but that the alterations to sub-cellular organization and cell carbohydrate status cannot be completely avoided using this strategy.

Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization.

A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC-ESI-MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performed using L-norvaline as standard. A limit of detection as low as 1 fmol/µl with a linear range of up to 125 pmol/µl could be obtained. Intraday and interday precisions were lower than 10 % relative standard deviations for most of the amino acids. Quantification using L-norvaline as internal standard gave very similar results compared to the quantification using deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 µm).

Kinetic analysis of Arabidopsis glucosyltransferase UGT74B1 illustrates a general mechanism by which enzymes can escape product inhibition.

Plant genomes encode numerous small molecule glycosyltransferases which modulate the solubility, activity, immunogenicity and/or reactivity of hormones, xenobiotics and natural products. The products of these enzymes can accumulate to very high concentrations, yet somehow avoid inhibiting their own biosynthesis. Glucosyltransferase UGT74B1 (UDP-glycosyltransferase 74B1) catalyses the penultimate step in the core biosynthetic pathway of glucosinolates, a group of natural products with important functions in plant defence against pests and pathogens. We found that mutation of the highly conserved Ser284 to leucine [wei9-1 (weak ethylene insensitive)] caused only very mild morphological and metabolic phenotypes, in dramatic contrast with knockout mutants, indicating that steady state glucosinolate levels are actively regulated even in unchallenged plants. Analysis of the effects of the mutation via a structural modelling approach indicated that the affected serine interacts directly with UDP-glucose, but also predicted alterations in acceptor substrate affinity and the kcat value, sparking an interest in the kinetic behaviour of the wild-type enzyme. Initial velocity and inhibition studies revealed that UGT74B1 is not inhibited by its glycoside product. Together with the effects of the missense mutation, these findings are most consistent with a partial rapid equilibrium ordered mechanism. This model explains the lack of product inhibition observed both in vitro and in vivo, illustrating a general mechanism whereby enzymes can continue to function even at very high product/precursor ratios.

ER-resident proteins PDR2 and LPR1 mediate the developmental response of root meristems to phosphate availability.

Inadequate availability of inorganic phosphate (Pi) in the rhizosphere is a common challenge to plants, which activate metabolic and developmental responses to maximize Pi acquisition. The sensory mechanisms that monitor environmental Pi status and regulate root growth via altered meristem activity are unknown. Here, we show that PHOSPHATE DEFICIENCY RESPONSE 2 (PDR2) encodes the single P(5)-type ATPase of Arabidopsis thaliana. PDR2 functions in the endoplasmic reticulum (ER) and is required for proper expression of SCARECROW (SCR), a key regulator of root patterning, and for stem-cell maintenance in Pi-deprived roots. We further show that the multicopper oxidase encoded by LOW PHOSPHATE ROOT 1 (LPR1) is targeted to the ER and that LPR1 and PDR2 interact genetically. Because the expression domains of both genes overlap in the stem-cell niche and distal root meristem, we propose that PDR2 and LPR1 function together in an ER-resident pathway that adjusts root meristem activity to external Pi. Our data indicate that the Pi-conditional root phenotype of pdr2 is not caused by increased Fe availability in low Pi; however, Fe homeostasis modifies the developmental response of root meristems to Pi availability.

Bacterial-type ferroxidase tunes iron-dependent phosphate sensing during Arabidopsis root development.

Access to inorganic phosphate (Pi), a principal intermediate of energy and nucleotide metabolism, profoundly affects cellular activities and plant performance. In most soils, antagonistic Pi-metal interactions restrict Pi bioavailability, which guides local root development to maximize Pi interception. Growing root tips scout the essential but immobile mineral nutrient; however, the mechanisms monitoring external Pi status are unknown. Here, we show that Arabidopsis LOW PHOSPHATE ROOT 1 (LPR1), one key determinant of Fe-dependent Pi sensing in root meristems, encodes a novel ferroxidase of high substrate specificity and affinity (apparent KM ∼ 2 µM Fe2+). LPR1 typifies an ancient, Fe-oxidizing multicopper protein family that evolved early upon bacterial land colonization. The ancestor of streptophyte algae and embryophytes (land plants) acquired LPR1-type ferroxidase from soil bacteria via horizontal gene transfer, a hypothesis supported by phylogenomics, homology modeling, and biochemistry. Our molecular and kinetic data on LPR1 regulation indicate that Pi-dependent Fe substrate availability determines LPR1 activity and function. Guided by the metabolic lifestyle of extant sister bacterial genera, we propose that Arabidopsis LPR1 monitors subtle concentration differentials of external Fe availability as a Pi-dependent cue to adjust root meristem maintenance via Fe redox signaling and cell wall modification. We further hypothesize that the acquisition of bacterial LPR1-type ferroxidase by embryophyte progenitors facilitated the evolution of local Pi sensing and acquisition during plant terrestrialization.

Chemoenzymatic synthesis of diverse thiohydroximates from glucosinolate-utilizing enzymes from Helix pomatia and Caldicellulosiruptor saccharolyticus.

Thiohydroximates comprise a diverse class of compounds important in both biological and industrial chemistry. Their syntheses are generally limited to simple alkyl and aryl compounds with few stereocenters and a narrow range of functional groups. We hypothesized that sequential action of two recombinant enzymes, a sulfatase from Helix pomatia and a ß-O-glucosidase from Caldicellulosiruptor saccharolyticus, on glucosinolates would allow synthesis of thiohydroximates from a structurally broad array of abundant precursors. We report successful synthesis of thiohydroximates of varied chemical classes, including from homochiral compounds of demonstrated biological activity. The chemoenzymatic synthetic route reported here should allow access to many, if not all, of the thiohydroximate core structures of the ~200 known naturally occurring glucosinolates. The enrichment of this group for compounds with possible pharmacological potential is discussed.

IQ67 DOMAIN proteins facilitate preprophase band formation and division-plane orientation.

Spatiotemporal control of cell division is essential for the growth and development of multicellular organisms. In plant cells, proper cell plate insertion during cytokinesis relies on the premitotic establishment of the division plane at the cell cortex. Two plant-specific cytoskeleton arrays, the preprophase band (PPB) and the phragmoplast, play important roles in division-plane orientation and cell plate formation, respectively1. Microtubule organization and dynamics and their communication with membranes at the cortex and cell plate are coordinated by multiple, mostly distinct microtubule-associated proteins2. How division-plane selection and establishment are linked, however, is still unknown. Here, we report members of the Arabidopsis IQ67 DOMAIN (IQD) family3 as microtubule-targeted proteins that localize to the PPB and phragmoplast and additionally reside at the cell plate and a polarized cortical region including the cortical division zone (CDZ). IQDs physically interact with PHRAGMOPLAST ORIENTING KINESIN (POK) proteins4,5 and PLECKSTRIN HOMOLOGY GTPase ACTIVATING (PHGAP) proteins6, which are core components of the CDZ1. The loss of IQD function impairs PPB formation and affects CDZ recruitment of POKs and PHGAPs, resulting in division-plane positioning defects. We propose that IQDs act as cellular scaffolds that facilitate PPB formation and CDZ set-up during symmetric cell division.