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Large-scale identification of genes expressed in roots of the model plant Arabidopsis was performed by serial analysis of gene expression (SAGE), on a total of 144,083 sequenced tags, representing at least 15,964 different mRNAs. For tag to gene assignment, we developed a computational approach based on 26,620 genes annotated from the complete sequence of the genome. The procedure selected warrants the identification of the genes corresponding to the majority of the tags found experimentally, with a high level of reliability, and provides a reference database for SAGE studies in Arabidopsis. This new resource allowed us to characterize the expression of more than 3,000 genes, for which there is no expressed sequence tag (EST) or cDNA in the databases. Moreover, 85% of the tags were specific for one gene. To illustrate this advantage of SAGE for functional genomics, we show that our data allow an unambiguous analysis of most of the individual genes belonging to 12 different ion transporter multigene families. These results indicate that, compared with EST-based tag to gene assignment, the use of the annotated genome sequence greatly improves gene identification in SAGE studies. However, more than 6,000 different tags remained with no gene match, suggesting that a significant proportion of transcripts present in the roots originate from yet unknown or wrongly annotated genes. The root transcriptome characterized in this study markedly differs from those obtained in other organs, and provides a unique resource for investigating the functional specificities of the root system. As an example of the use of SAGE for transcript profiling in Arabidopsis, we report here the identification of 270 genes differentially expressed between roots of plants grown either with NO[sub 3, sup -] or NH[sub 4]NO[sub 3] as N source. [ABSTRACT FROM AUTHOR]

In Arabidopsis thaliana , root high-affinity nitrate (NO3–) uptake depends mainly on NRT2.1, 2.4, and 2.5, which are repressed by high NO3– supply at the transcript level. For NRT2.1 , this regulation is due to the action of (i) feedback down-regulation by N metabolites and (ii) repression by NO3– itself mediated by the transceptor NRT1.1(NPF6.3). However, for NRT2.4 and NRT2.5 , the signalling pathway(s) remain unknown as do the molecular elements involved. Here we show that unlike NRT2.1 , NRT2.4 and NRT2.5 are not induced in an NO3– reductase mutant but are up-regulated following replacement of NO3– by ammonium (NH4+) as the N source. Moreover, increasing the NO3– concentration in a mixed nutrient solution with constant NH4+ concentration results in a gradual repression of NRT2.4 and NRT2.5 , which is suppressed in an nrt1.1 mutant. This indicates that NRT2.4 and NRT2.5 are subjected to repression by NRT1.1-mediated NO3– sensing, and not to feedback repression by reduced N metabolites. We further show that key regulators of NRT2 transporters, such as HHO1, HRS1, PP2C, LBD39, BT1, and BT2, are also regulated by NRT1.1-mediated NO3– sensing, and that several of them are involved in NO3– repression of NRT2.1 , NRT2.4 , and NRT2.5. Finally, we provide evidence that it is the phosphorylated form of NRT1.1 at the T101 residue, which is most active in triggering the NRT1.1-mediated NO3– regulation of all these genes. Altogether, these data led us to propose a regulatory model for high-affinity NO3– uptake in Arabidopsis, highlighting several NO3– transduction cascades downstream of the phosphorylated form of the NRT1.1 transceptor. [ABSTRACT FROM AUTHOR]

Diazotrophic bacteria isolated from the rhizosphere of a wild wheat ancestor, grown from its refuge area in the Fertile Crescent, were found to be efficient Plant Growth-Promoting Rhizobacteria (PGPR), upon interaction with an elite wheat cultivar. In nitrogen-starved plants, they increased the amount of nitrogen in the seed crop (per plant) by about twofold. A bacterial growth medium was developed to investigate the effects of bacterial exudates on root development in the elite cultivar, and to analyze the exo-metabolomes and exo-proteomes. Altered root development was observed, with distinct responses depending on the strain, for instance, with respect to root hair development. A first conclusion from these results is that the ability of wheat to establish effective beneficial interactions with PGPRs does not appear to have undergone systematic deep reprogramming during domestication. Exo-metabolome analysis revealed a complex set of secondary metabolites, including nutrient ion chelators, cyclopeptides that could act as phytohormone mimetics, and quorum sensing molecules having inter-kingdom signaling properties. The exo-proteome-comprised strain-specific enzymes, and structural proteins belonging to outer-membrane vesicles, are likely to sequester metabolites in their lumen. Thus, the methodological processes we have developed to collect and analyze bacterial exudates have revealed that PGPRs constitutively exude a highly complex set of metabolites; this is likely to allow numerous mechanisms to simultaneously contribute to plant growth promotion, and thereby to also broaden the spectra of plant genotypes (species and accessions/cultivars) with which beneficial interactions can occur. [ABSTRACT FROM AUTHOR]

Polycomb-group (PcG) proteins are major chromatin complexes that regulate gene expression, mainly described as repressors keeping genes in a transcriptionally silent state during development. Recent studies have nonetheless suggested that PcG proteins might have additional functions, including targeting active genes or acting independently of gene expression regulation. However, the reasons for the implication of PcG proteins and their associated chromatin marks on active genes are still largely unknown. Here, we report that combining mutations for CURLY LEAF (CLF) and LIKE HETEROCHROMATIN PROTEIN1 (LHP1), two Arabidopsis PcG proteins, results in deregulation of expression of active genes that are targeted by PcG proteins or enriched in associated chromatin marks. We show that this deregulation is associated with accumulation of small RNAs corresponding to massive degradation of active gene transcripts. We demonstrate that transcriptionally active genes and especially those targeted by PcG proteins are prone to RNA degradation, even though deregulation of RNA degradation following the loss of function of PcG proteins is not likely to be mediated by a PcG protein-mediated chromatin environment. Therefore, we conclude that PcG protein function is essential to maintain an accurate level of RNA degradation to ensure accurate gene expression. [ABSTRACT FROM AUTHOR]

Plant defensins are recognized for their antifungal properties. However, a few type 1 defensins ( PDF1s) were identified for their cellular zinc (Zn) tolerance properties after a study of the metal extremophile Arabidopsis halleri. In order to investigate whether different paralogues would display specialized functions, the A. halleri PDF1 family was characterized at the functional and genomic levels., Eleven PDF1s were isolated from A. halleri. Their ability to provide Zn tolerance in yeast cells, their activity against Fusarium oxysporum f. sp. melonii, and their level of expression in planta were compared with those of the seven A. thaliana PDF1s. The genomic organization of the PDF1 family was comparatively analysed within the Arabidopsis genus., Ah PDF1s and At PDF1s were able to confer Zn tolerance and Ah PDF1s also displayed antifungal activity. PDF1 transcripts were constitutively more abundant in A. halleri than in A. thaliana. Within the Arabidopsis genus, the PDF1 family is evolutionarily dynamic, in terms of gain and loss of gene copy., Arabidopsis halleri PDF1s display no superior abilities to provide Zn tolerance. A constitutive increase in Ah PDF1 transcript accumulation is proposed to be an evolutionary innovation co-opting the promiscuous PDF1 protein for its contribution to Zn tolerance in A. halleri. [ABSTRACT FROM AUTHOR]

Methylation of biologically active molecules is achieved by methyltransferases (MTases). MTases can act on proteins through N- or O-carboxylmethylation reactions. Methylation of lysine and glutamic acid residues was recently described on the N-terminal tail of AtPIP2;1, a plasma membrane aquaporin of plants. In this study, we combine a bioinformatic and a biochemical screen and identify two MTases of Arabidopsis thaliana, SDG7 (At2g44150) and OMTF3 (At3g61990), as acting on the N-terminal tail of AtPIP2;1, at Lys3 and Glu6, respectively. Confocal microscopy imaging showed the two enzymes to be associated with the endoplasmic reticulum. An in vitro assay using various AtPIP2;1 N-terminal peptides as a bait allowed characterization of the enzymatic properties of recombinant SDG7 and OMTF3. The two enzymes showed minimal apparent Km values for their substrates, S-adenosylmethionine and peptide, in the range of 5–8 and 2–9 μM, respectively. SDG7 was shown to almost exclusively mono- or di-methylate Lys3. In contrast, OMTF3 specifically methylated Glu6, this methylation being dependent on the methylation profile of the neighboring Lys3 residue. In conclusion, this study allows the characterization of the first MTases able to methylate plant transmembrane proteins and provides the first identification of a glutamate-MTase in eukaryotes. [ABSTRACT FROM AUTHOR]

Amongst the nine voltage-gated K+ channel (Kv) subunits expressed in Arabidopsis, AtKC1 does not seem to form functional Kv channels on its own, and is therefore said to be silent. It has been proposed to be a regulatory subunit, and to significantly influence the functional properties of heteromeric channels in which it participates, along with other Kv channel subunits. The mechanisms underlying these properties of AtKC1 remain unknown. Here, the transient (co-)expression of AtKC1, AKT1 and/or KAT1 genes was obtained in tobacco mesophyll protoplasts, which lack endogenous inward Kv channel activity. Our experimental conditions allowed both localization of expressed polypeptides (GFP-tagging) and recording of heterologously expressed Kv channel activity (untagged polypeptides). It is shown that AtKC1 remains in the endoplasmic reticulum unless it is co-expressed with AKT1. In these conditions heteromeric AtKC1-AKT1 channels are obtained, and display functional properties different from those of homomeric AKT1 channels in the same context. In particular, the activation threshold voltage of the former channels is more negative than that of the latter ones. Also, it is proposed that AtKC1-AKT1 heterodimers are preferred to AKT1-AKT1 homodimers during the process of tetramer assembly. Similar results are obtained upon co-expression of AtKC1 with KAT1. The whole set of data provides evidence that AtKC1 is a conditionally-targeted Kv subunit, which probably downregulates the physiological activity of other Kv channel subunits in Arabidopsis. [ABSTRACT FROM AUTHOR]

Arabidopsis thaliana mutants deficient for the NRTI.1 NO3- transporter display complex phenotypes, including lowered NO3- uptake, altered development of nascent organs, and reduced stomatal opening. To obtain further insight at the molecular level on the multiple physiological functions of NRT1.1, we performed large-scale transcript profiling by serial analysis of gene expression in the roots of the chl1-5 deletion mutant of NRT1.1 and of the Columbia wild type. Several hundred genes were differentially expressed between the two genotypes, when plants were grown on NH4NO3 as N source. Among these genes, the N satiety-repressed NRT2.1 gene, encoding a major component of the root high-affinity NO3-transport system (HATS), was found to be strongly derepressed in the chl1-5 mutant (as well as in other NRT1.1 mutants). This was associated with a marked stimulation of the NO3- HATS activity in the mutant, suggesting adaptive response to a possible N limitation resulting from NRT1.1 mutation. However, derepression of NRT2.1 in NH4NO3-fed chl1-5 plants could not be attributed to lowered production of N metabolites. Rather, the results show that normal regulation of NRT2.1 expression is strongly altered in the chl1-5 mutant, where this gene is no more repressible by high N provision to the plant. This indicates that NRT1.1 plays an unexpected but important role in the regulation of both NRT2.1 expression and NO3-HATS activity. Overexpression of NRT2.1 was also found in wild-type plants supplied with 1 mM NH4+ plus 0.1 mM NO3-, a situation where NRT1.1 is likely to mediate very low NO3- transport. Thus, we suggest that it is the lack of NRT1.1 activity, rather than the absence of this transporter, that derepresses NRT2.1 expression in the presence of NH4+. Two hypotheses are discussed to explain these results: (1) NRT2.1... [ABSTRACT FROM AUTHOR]

In Arabidopsis (Arabidopsis thaliana), the High-Affinity Transport System (HATS) for root nitrate (NO3-) uptake depends mainly on four NRT2 NO-3 transporters, namely NRT2.1, NRT2.2, NRT2.4, and NRT2.5. The HATS is the target of many regulations to coordinate nitrogen (N) acquisition with the N status of the plant and with carbon (C) assimilation through photosynthesis. At the molecular level, C and N signaling pathways control gene expression of the NRT2 transporters. Although several regulators of these transporters have been identified in response to either N or C signals, the response of NRT2 gene expression to the interaction of these signals has never been specifically investigated, and the underlying molecular mechanisms remain largely unknown. To address this question we used an original systems biology approach to model a regulatory gene network targeting NRT2.1, NRT2.2, NRT2.4, and NRT2.5 in response to N/C signals. Our systems analysis of the data identified three transcription factors, TGA3, MYC1, and bHLH093. Functional analysis of mutants combined with yeast one-hybrid experiments confirmed that all three transcription factors are regulators of NRT2.4 or NRT2.5 in response to N or C signals. These results reveal a role for TGA3, MYC1, and bHLH093 in controlling the expression of root NRT2 transporter genes. [ABSTRACT FROM AUTHOR]

Potassium (K+) acquisition, translocation and cellular homeostasis are mediated by various membrane transport systems in all organisms. We identified and described an ion channel in the ectomycorrhizal fungus Hebeloma cylindrosporum (HcSKC) that harbors features of animal voltage-dependent Shaker-like K+ channels, and investigated its role in both free-living hyphae and symbiotic conditions. RNAi lines affected in the expression of HcSKC were produced and used for in vitro mycorrhizal assays with the maritime pine as host plant, under standard or low K+ conditions. The adaptation of H. cylindrosporum to the downregulation of HcSKC was analyzed by qRT-PCR analyses for other K+-related transport proteins: the transporters HcTrk1, HcTrk2, and HcHAK, and the ion channels HcTOK1, HcTOK2.1, and HcTOK2.2. Downregulated HcSKC transformants displayed greater K+ contents at standard K+ only. In such conditions, plants inoculated with these transgenic lines were impaired in K+ nutrition. Taken together, these results support the hypothesis that the reduced expression of HcSKC modifies the pool of fungal K+ available for the plant and/or affects its symbiotic transfer to the roots. Our study reveals that the maintenance of K+ transport in H. cylindrosporum, through the regulation of HcSKC expression, is required for the K+ nutrition of the host plant. [ABSTRACT FROM AUTHOR]

Summary: In Arabidopsis thaliana, NRT2.1 codes for a main component of the root nitrate high‐affinity transport system. Previous studies revealed that post‐translational regulation of NRT2.1 plays an important role in the control of root nitrate uptake and that one mechanism could correspond to NRT2.1 C‐terminus processing.To further investigate this hypothesis, we produced transgenic plants with truncated forms of NRT2.1. This revealed an essential sequence for NRT2.1 activity, located between the residues 494 and 513.Using a phospho‐proteomic approach, we found that this sequence contains one phosphorylation site, at serine 501, which can inactivate NRT2.1 function when mimicking the constitutive phosphorylation of this residue in transgenic plants. This phenotype could neither be explained by changes in abundance of NRT2.1 and NAR2.1, a partner protein of NRT2.1, nor by a lack of interaction between these two proteins.Finally, the relative level of serine 501 phosphorylation was found to be increased by ammonium nitrate in wild‐type plants, leading to the inactivation of NRT2.1 and to a decrease in high affinity nitrate transport into roots. Altogether, these observations reveal a new and essential mechanism for the regulation of NRT2.1 activity. See also the Commentary on this article by Rahikainen & Kangasjärvi (2020), 228: 802–804. [ABSTRACT FROM AUTHOR]

Control of K+ and Na+ transport plays a central role in plant adaptation to salinity. In the halophyte Hordeum maritimum , we have characterized a transporter gene, named HmHKT2;1 , whose homolog HvHKT2;1 in cultivated barley, Hordeum vulgare , was known to give rise to increased salt tolerance when overexpressed. The encoded protein is strictly identical in two H. maritimum ecotypes, from two biotopes (Tunisian sebkhas) affected by different levels of salinity. These two ecotypes were found to display distinctive responses to salt stress in terms of biomass production, Na+ contents, K+ contents and K+ absorption efficiency. Electrophysiological analysis of HmHKT2;1 in Xenopus oocytes revealed distinctive properties when compared with HvHKT2;1 and other transporters from the same group, especially a much higher affinity for both Na+ and K+, and an Na+–K+ symporter behavior in a very broad range of Na+ and K+ concentrations, due to reduced K+ blockage of the transport pathway. Domain swapping experiments identified the region including the fifth transmembrane segment and the adjacent extracellular loop as playing a major role in the determination of the affinity for Na+ and the level of K+ blockage in these HKT2;1 transporters. The analysis (quantitative reverse transcription-PCR; qRT-PCR) of HmHKT2;1 expression in the two ecotypes submitted to saline conditions revealed that the levels of HmHKT2;1 transcripts were maintained constant in the most salt-tolerant ecotype whereas they decreased in the less tolerant one. Both the unique functional properties of HmHKT2;1 and the regulation of the expression of the encoding gene could contribute to H. maritimum adaptation to salinity. [ABSTRACT FROM AUTHOR]

Abstract: Cloning and characterizations of plant K+ transport systems aside from Arabidopsis have been increasing over the past decade, favored by the availability of more and more plant genome sequences. Information now available enables the comparison of some of these systems between species. In this review, we focus on three families of plant K+ transport systems that are active at the plasma membrane: the Shaker K+ channel family, comprised of voltage-gated channels that dominate the plasma membrane conductance to K+ in most environmental conditions, and two families of transporters, the HAK/KUP/KT K+ transporter family, which includes some high-affinity transporters, and the HKT K+ and/or Na+ transporter family, in which K+-permeable members seem to be present in monocots only. The three families are briefly described, giving insights into the structure of their members and on functional properties and their roles in Arabidopsis or rice. The structure of the three families is then compared between plant species through phylogenic analyses. Within clusters of ortologues/paralogues, similarities and differences in terms of expression pattern, functional properties and, when known, regulatory interacting partners, are highlighted. The question of the physiological significance of highlighted differences is also addressed. [Copyright &y& Elsevier]

Zinc (Zn) is essential for the optimal growth of plants but is toxic if present in excess, so Zn homeostasis needs to be finely tuned. Understanding Zn homeostasis mechanisms in plants will help in the development of innovative approaches for the phytoremediation of Zn-contaminated sites. In this study, Zn tolerance quantitative trait loci (QTL) were identified by analyzing differences in the Bay-0 and Shahdara accessions of Arabidopsis thaliana. Fine-scale mapping showed that a variant of the Fe homeostasis-related FERRIC REDUCTASE DEFECTIVE3 (FRD3) gene, which encodes a multidrug and toxin efflux (MATE) transporter, is responsible for reduced Zn tolerance in A. thaliana. Allelic variation in FRD3 revealed which amino acids are necessary for FRD3 function. In addition, the results of allele-specific expression assays in F1 individuals provide evidence for the existence of at least one putative metal-responsive cis-regulatory element. Our results suggest that FRD3 works as a multimer and is involved in loading Zn into xylem. Cross-homeostasis between Fe and Zn therefore appears to be important for Zn tolerance in A. thaliana with FRD3 acting as an essential regulator. [ABSTRACT FROM AUTHOR]

Zinc (Zn) is essential for the optimal growth of plants but is toxic if present in excess, so Zn homeostasis needs to be finely tuned. Understanding Zn homeostasis mechanisms in plants will help in the development of innovative approaches for the phytoremediation of Zn-contaminated sites. In this study, Zn tolerance quantitative trait loci (QTL) were identified by analyzing differences in the Bay-0 and Shahdara accessions of Arabidopsis thaliana. Fine-scale mapping showed that a variant of the Fe homeostasis-related FERRIC REDUCTASE DEFECTIVE3 (FRD3) gene, which encodes a multidrug and toxin efflux (MATE) transporter, is responsible for reduced Zn tolerance in A. thaliana. Allelic variation in FRD3 revealed which amino acids are necessary for FRD3 function. In addition, the results of allele-specific expression assays in F1 individuals provide evidence for the existence of at least one putative metal-responsive cis-regulatory element. Our results suggest that FRD3 works as a multimer and is involved in loading Zn into xylem. Cross-homeostasis between Fe and Zn therefore appears to be important for Zn tolerance in A. thaliana with FRD3 acting as an essential regulator. [ABSTRACT FROM AUTHOR]

Plant growth under low K+ availability or salt stress requires tight control of K+ and Na+ uptake, long-distance transport, and accumulation. The family of membrane transporters named HKT (for High-Affinity K+ Transporters), permeable either to K+ and Na+ or to Na+ only, is thought to play major roles in these functions. Whereas Arabidopsis (Arabidopsis thaliana) possesses a single HKT transporter, involved in Na+ transport in vascular tissues, a larger number of HKT transporters are present in rice (Oryza sativa) as well as in other monocots. Here, we report on the expression patterns and functional properties of three rice HKT transporters, OsHKTI;1, OsHKT1;3, and OsHKT2;1. In situ hybridization experiments revealed overlapping but distinctive and complex expression patterns, wider than expected for such a transporter type, including vascular tissues and root periphery but also new locations, such as osmocontractile leaf bulliform cells (involved in leaf folding). Functional analyses in Xenopus laevis oocytes revealed striking diversity. OsHKT1;1 and OsHKT1;3, shown to be permeable to Na+ only, are strongly different in terms of affinity for this cation and direction of transport (inward only or reversible). OsHKT2;1 displays diverse permeation modes, Na+-K+ symport, Na+ uniport, or inhibited states, depending on external Na+ and K+ concentrations within the physiological concentration range. The whole set of data indicates that HKT transporters fulfill distinctive roles at the whole plant level in rice, each system playing diverse roles in different cell types. Such a large diversity within the HKT transporter family might be central to the regulation of K+ and Na+ accumulation in monocots. [ABSTRACT FROM AUTHOR]

Abstract: This is the first report of a systematic study of genes expressed by means of expressed sequence tag (EST) analysis in oil palm, a species of the Arecales order, a phylogenetically key clade of monocotyledons that is not widely represented in the sequence databases. Five different cDNA libraries were generated from male and female inflorescences, shoot apices and zygotic embryos and unidirectional systematic sequencing was performed. A total of 2411 valid EST sequences were thus obtained. Cluster analysis enabled the identification of 209 groups of related sequences and 1874 singletons. Putative functions were assigned to 1252 of the set of 2083 non-redundant ESTs obtained. The EST database described here is a first step towards gene discovery and cDNA array-based expression analysis in oil palm. [Copyright &y& Elsevier]

• In an attempt to advance the understanding of ectomycorrhizal symbiosis, which improves mineral nutrition and resistance to abiotic and biotic stresses of most woody plants from temperate and boreal regions, we produced a cDNA library of the ectomycorrhizal fungusHebeloma cylindrosporumin a yeast expression vector. The library was used for functional complementation cloning and for sequencing of 4036 expressed sequence tags (ESTs).• Expression of the library in a yeastade2andtrp1mutant strain allowed cloning of twoHebelomagenes,HcADE2andHcTRP1by functional complementation. This experiment highlighted both the efficiency of the complementation strategy and the quality of the library.• All sequence data available onHebeloma(4596) were analysed and contiged. The whole EST resource is accessibleviaa web interface, which allows searching and navigation through clusters and tentative contigs (TCs).• Analysis of the database revealed a low level of counterparts already identified in other ectomycorrhizal fungi. For example, it allowed identification of a large set of membrane transporters, such as phosphate, potassium, sulphate or micronutrient transporters. Thus, this EST resource provides a valuable tool for research on ectomycorrhiza and symbiotic interactions.New Phytologist(2004)doi: 10.1111/j.1469-8137.2004.01185.x© New Phytologist(2004) [ABSTRACT FROM AUTHOR]

The number of genes in the human genome is unknown, with estimates ranging from 50,000 to 90,000 (refs 1,2), and to more than 140,000 according to unpublished sources. We have developed 'Exofish', a procedure based on homology searches, to identify human genes quickly and reliably. This method relies on the sequence of another vertebrate, the pufferfish Tetraodon nigroviridis, to detect conserved sequences with a very low background. Similar to Fugu rubripes, a marine pufferfish proposed by Brenner et al. as a model for genomic studies, T. nigroviridis is a more practical alternative with a genome also eight times more compact than that of human. Many comparisons have been made between F. rubripes and human DNA that demonstrate the potential of comparative genomics using the pufferfish genome. Application of Exofish to the December version of the working draft sequence of the human genome and to Unigene showed that the human genome contains 28,000-34,000 genes, and that Unigene contains less than 40% of the protein-coding fraction of the human genome. [ABSTRACT FROM AUTHOR]

Meleda disease (mal de Meleda) MIM *248300 is an autosomal recessive disorder, clinically characterised by transgressive palmoplantar keratoderma, hyperhidrosis and perioral erythema. It was first described on the Adriatic island of Meleda, where it was relatively common. The prevalance in the general population is estimated to be 1 in 100 000. Linkage analysis of two large consanguineous families from Algeria, including 10 affected individuals, showed strong evidence for localisation of Meleda disease to chromosome 8qter with a maximum two-point lod score for D8S1751 of 8.21 at Θ = 0. Analysis of homozygosity regions and recombination events places the the gene in a region of at least 3 cM, telomeric to D8S1727. A common haplotype was observed in the two families, suggesting a founder effect. [ABSTRACT FROM AUTHOR]