Salt tolerance is a commercially important trait that affects plant species around the globe. Cellular response to saline environments is a well studied but complex system that is far from being completely understood. The SALT OVERLY SENSITIVE 1 (SOS1) gene is a critical component of salt tolerance in many species, encoding a plasma membrane Na+/H+ antiporter that plays an important role in germination and growth in saline environments. Here we report a preliminary investigation of salt tolerance in quinoa (Chenopodium quinoa Willd.). Quinoa is a halophytic grain crop of the Chenopodiaceae family with impressive nutritional content and an increasing world-wide market. Many quinoa varieties have impressive salt tolerance characteristics and research suggests quinoa may utilize novel mechanisms to confer salt tolerance. At this time there is no published data on the molecular characteristics of those mechanisms. We report the identification and sequencing of the SOS1 gene in quinoa, including a full length cDNA sequence of 3490 bp and a full length genomic clone of 21314 bp. Sequence analysis predicts the quinoa SOS1 homolog spans 23 exons and is comprised of 3474 bp of coding sequence (excluding the stop codon). Introns comprise 17840 bp of the genomic clone and range in size from 77 to 2123 bp. The predicted protein contains 1158 amino acid residues and aligns closely with SOS1 homologs of other species. The quinoa SOS1 homolog contains two putative domains, a Nhap cation-antiporter domain and a cyclic-nucleotide binding domain. Sequence analyses of both cDNA fragments and intron fragments suggest that two SOS1 loci are present in the quinoa genome that are likely orthologous loci derived from the ancestral diploid genomes of the modern allotetraploid quinoa genome. This report represents the first molecular characterization of a putative salt-tolerance gene in C. quinoa.

The accumulation of salts in soil is an important agricultural problem that limits crop productivity. Salts containing sodium (Na⁺) are particularly problematic, as cytosolic Na⁺ can interfere with cellular metabolism and lead to cell death. Maintaining low levels of cytosolic Na⁺, therefore, is critical for plant survival during growth in salt. Mechanisms to regulate Na⁺ accumulation in plant cells include extrusion of Na⁺ from the cell and sequestration of Na⁺ into intracellular compartments. Both of these processes are controlled in part through the action of Na⁺/H⁺ exchangers belonging to the Cation/Proton Antiporter-1 (CPA1) gene family. Genes belonging to this family have been identified in both salt-sensitive and salt-tolerant species, suggesting that salt-tolerant species may have evolved salt tolerance through modification of these existing pathways. The research presented here has focused on understanding how salt tolerance has evolved in Brassicaceae species, and particularly on the role that CPA1 genes have played in the adaptation to salinity of Eutrema salsugineum. Specific projects have sought to understand 1) how copy number variation and changes in coding sequences of CPA1 genes contribute to salt tolerance in E. salsugineum and its salt-tolerant relative Schrenkiella parvula, 2) whether functional or regulatory changes in Salt Overly Sensitive 1 (SOS1) from E. salsugineum (EsSOS1) contribute to its enhanced salt tolerance, and 3) whether accessions of Arabidopsis thaliana differ significantly in their response to salt stress.The results indicate that EsSOS1 and SOS1 from S. parvula (SpSOS1) both confer greater salt tolerance in yeast than SOS1 from A. thaliana (AtSOS1) when activated by the complex of the SOS2 kinase and SOS3 calcium-binding protein, whereas only EsSOS1 confers enhanced salt tolerance in the absence of activation. When expressed in A. thaliana, EsSOS1 also confers greater salt tolerance than AtSOS1 through regulatory changes that likely involve differences in expression pattern. Together, the results presented here suggest that mechanisms regulating cellular Na⁺ accumulation that exist in salt-sensitive crop species could be altered to enhance growth in salty soils. In addition, the 19 A. thaliana accessions used to create the MAGIC population were shown to differ significantly in their response to salt stress.

TORQUATO, José Pedro Pires. Estudo de mecanismos de regulação do sódio citoplasmático em cultivares de Vigna unguiculata (L.) WALP. submetidas à salinidade. 2014. 139 f. Tese (Doutorado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2014. / Submitted by Anderson Silva Pereira (anderson.pereiraaa@gmail.com) on 2017-01-23T20:58:55Z No. of bitstreams: 1 2016_dis_jpptorquato.pdf: 3572903 bytes, checksum: be55e53ccae4d0b5e66f15e6780ce1c8 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-01-23T21:20:32Z (GMT) No. of bitstreams: 1 2016_dis_jpptorquato.pdf: 3572903 bytes, checksum: be55e53ccae4d0b5e66f15e6780ce1c8 (MD5) / Made available in DSpace on 2017-01-23T21:20:32Z (GMT). No. of bitstreams: 1 2016_dis_jpptorquato.pdf: 3572903 bytes, checksum: be55e53ccae4d0b5e66f15e6780ce1c8 (MD5) Previous issue date: 2014 / In the Northeast of Brazil, there are some salinized areas and others in the process of salinization, what drastically reduces plant productivity. Many studies have been done regarding the effect of saline stress on plant growth and development. However, there is little knowledge about the mechanisms of acclimatization to salinity in the different plant organs, particularly in the maintenance mechanisms of sodium ion homeostasis in Vigna unguiculata, involving: enzymes, channels and transporters. For this, two cultivars with different degrees of susceptibility to saline stress were used: Pitiúba (tolerant) and Setentão (sensitive). The objective of the present work was to understand the interrelationships between the H+-ATPases and the SOS1 transporter, as well as the interrelationships between the vacuolar NHX transporter and the vacuolar proton pumps and their roles on regulating the Na+ cytoplasmic concentration in Vigna unguiculata, in response to saline stress. For this, morphological parameters were evaluated: roots, stems and leaves length, root, stems and leaves dry mass, ratio Root/Aerial, and biochemical parameters: proline, malondialdehyde, Na+ and K+ ions and the ratio Na+/K+, at 5 different concentrations (0, 25, 50, 75 and 100 mM) of NaCl. Molecular parameters in the two cultivars Pitiúba and Setentão were also analyzed in plants with 10 days after sowing, submitted to NaCl (100 mM) for 120 hours. SOS1, H+-ATPase type P and type V, pyrophosphatases and NHX encoding genes were identified through searches in Vigna unguiculata databases. The gene expression was evaluated in plants with 10 days after sowing, submitted to 100 mM NaCl, at the times: 0, 6, 12, 24 and 48 hours after stress application, using PCR technique in real-time (qPCR). The entirely evaluation of the data allowed to establish the differences in the form of acclimatization to the saline stress between the two cultivars. The parameters such as visual aspects, dry mass, leaf area and Na+/K+ ratio gave support for Pitiúba to be classified as a salt tolerant cultivar in relation to the Setentão. The parameters, growth and percentage of water were not conclusive. The Pitiúba cultivar, presented a constitutive proline concentration in the leaves greater than the Setentão cultivar and being, therefore, better pre-acclimatized to tolerate saline stress. The highest lipid peroxidation in the leaves was observed in Pitiúba. The database searches revealed that V. unguiculata presents at least 1 SOS1 gene, 13 H+-ATPase type P genes, 7 NHX genes, 2 PPase genes and 1 V-ATPase subA gene. Of these genes, SOS1, H+-ATPase 06 and 48, NHX1, NHX2, PPase1 and V-ATPase subA were induced in response to saline stress in a differentiated way in relation to organ and cultivar analyzed. From these data it was possible to infer that the exclusion of Na+ in Pitiúba occurs mainly in the roots, while in the Setentão it occurs more intensely in the leaves. Vacuolar compartmentalization of Na+ is higher in the roots of both cultivars. However, while Pitiúba increased expression of NHX1, Setentão increased expression of NHX2. NHX induction was accompanied by the mRNA increase of PPase1 in both cultivars and V-ATPase in Pitiúba. In leaves, the SOS1 gene was most expressed in the Setentão, revealing a co-expression with two H+-ATPase genes (H+-ATPase 06 and 48), whereas NHX1 expression increased only in Pitiúba. In general, the data suggest that the cultivars of Vigna unguiculata (Pitiúba and Setentão) used different strategies to minimize the deleterious effects of saline stress. / No Nordeste brasileiro existem algumas áreas salinizadas e outras em processo de salinização, o que reduz drasticamente a produtividade das plantas. Muitos estudos tem sido feitos em relação ao efeito do estresse salino no crescimento e desenvolvimento das plantas. Contudo, pouco se sabe sobre os mecanismos de aclimatação à salinidade nos diferentes órgãos vegetais, particularmente nos mecanismos de manutenção da homeostase iônica do sódio em Vigna unguiculata, envolvendo: enzimas, canais e transportadores. Para tal, foram utilizados dois cultivares com diferentes graus de suscetibilidade ao estresse salino: Pitiúba (tolerante) e Setentão (sensível). O objetivo do presente trabalho foi compreender as inter-relações entre as H+-ATPases e o transportador SOS1, bem como, as inter-relações entre o transportador vacuolar NHX e as bombas de prótons vacuolares e seus papéis na regulação da concentração citoplasmática de Na+ em Vigna unguiculata, em resposta ao estresse salino. Para tal, foram avaliados parâmetros morfológicos: comprimento de raízes, caules e folhas, massa seca de raízes, caules e folhas, razão Raiz/Parte Aérea, e parâmetros bioquímicos: concentrações de prolina, malondialdeído, dos íons Na+ e K+ e a razão Na+/K+, em 5 diferentes concentrações (0, 25, 50, 75 e 100 mM) de NaCl. Também foram analisados parâmetros moleculares nos dois cultivares Pitiúba e Setentão em plantas com 10 dias após a semeadura, submetidas ao NaCl (100 mM) por 120 horas. Genes codificadores da SOS1, H+-ATPase tipo P e tipo V, pirofosfatases e NHX foram identificados através de buscas em bancos de dados de V. unguiculata. A expressão gênica foi avaliada em plantas com 10 dias após a semeadura, submetidas a NaCl 100 mM, nos tempos: 0, 6, 12, 24 e 48 horas após aplicação do estresse, através da técnica PCR em tempo real (qPCR). A avaliação conjunta dos dados possibilitou estabelecer as diferenças na forma de aclimatação ao estresse salino entre os dois cultivares. Os parâmetros tais como: aspectos visuais, massa seca, área foliar e razão Na+/K+ deram suporte para que Pitiúba sejá classificada como um cultivar mais tolerante ao estresse salino em relação a Setentão. Os parâmetros, crescimento e percentual de água não foram conclusivos. O cultivar Pitiúba, apresentou uma concentração constitutiva de prolina maior do que Setentão nas folhas estando, portanto, melhor pré-aclimatado para tolerância ao estresse salino. A maior peroxidação lipídica nas folhas foi observada em Pitiúba. As buscas em bancos de dados revelaram que V. unguiculata apresenta pelo menos 1 gene SOS1, 13 genes H+-ATPase tipo P, 7 genes NHX, 2 genes PPase e 1 gene V-ATPase subA. Desses genes, SOS1, H+-ATPase 06 e 48, NHX1, NHX2, PPase1 e V-ATPase subA foram induzidos em resposta ao estresse salino de maneira diferenciada em relação ao órgão e cultivar analisado. A partir desses dados foi possível inferir que a exclusão de Na+ em Pitiúba ocorre principalmente nas raízes, enquanto que em Setentão ocorre mais intensamente nas folhas. A compartimentalização vacuolar do Na+ é maior nas raízes de ambos os cultivares. Contudo, enquanto Pitiúba aumentou a expressão de NHX1, Setentão aumentou a expressão de NHX2. A indução de NHX foi acompanhada pelo aumento do mRNA da PPase1 em ambos os cultivares e da V-ATPase em Pitiúba. Em folhas, o gene SOS1 foi mais expresso em Setentão revelando uma co-expressão com dois genes da H+-ATPase (H+-ATPase 06 e 48), enquanto a expressão de NHX1 aumentou apenas em Pitiúba. Em geral, os dados sugerem que os cultivares de V. unguiculata (Pitiúba e Setentão) utilizaram diferentes estratégias para minimizar os efeitos deletérios do estresse salino.

Background: In Arabidopsis thaliana, the family of cyclic nucleotide-gated channels (CNGCs) is composed of 20 members. Previous studies indicate that plant CNGCs are involved in the control of growth processes and responses to abiotic and biotic stresses. According to their proposed function as cation entry pathways these channels contribute to cellular cation homeostasis, including calcium and sodium, as well as to stress-related signal transduction. Here, we studied the expression patterns and regulation of CNGC19 and CNGC20, which constitute one of the five CNGC subfamilies. Results: GUS, GFP and luciferase reporter assays were used to study the expression of CNGC19 and CNGC20 genes from Arabidopsis thaliana in response to developmental cues and salt stress. CNGC19 and CNGC20 were differentially expressed in roots and shoots. The CNGC19 gene was predominantly active in roots already at early growth stages. Major expression was observed in the phloem. CNGC20 showed highest promoter activity in mesophyll cells surrounding the veins. Its expression increased during development and was maximal in mature and senescent leaves. Both genes were upregulated in the shoot in response to elevated NaCl but not mannitol concentrations. While in the root, CNGC19 did not respond to changes in the salt concentration, in the shoot it was strongly upregulated in the observed time frame (6-72 hours). Salt-induction of CNGC20 was also observed in the shoot, starting already one hour after stress treatment. It occurred with similar kinetics, irrespective of whether NaCl was applied to roots of intact plants or to the petiole of detached leaves. No differences in K and Na contents of the shoots were measured in homozygous T-DNA insertion lines for CNGC19 and CNGC20, respectively, which developed a growth phenotype in the presence of up to 75 mM NaCl similar to that of the wild type. Conclusion: Together, the results strongly suggest that both channels are involved in the salinity response of different cell types in the shoot. Upon salinity both genes are upregulated within hours. CNGC19 and CNGC20 could assist the plant to cope with toxic effects caused by salt stress, probably by contributing to a re-allocation of sodium within the plant.

Control of intracellular ion homeostasis is pivotal to plant salt tolerance. Plants have developed a number of mechanisms to keep ions at appropriate concentrations. Both transporters and channels on the plasma membrane play important roles in this function. Plant cyclic nucleotide-gated channels (CNGCs) in the plasma membrane are non-selective monovalent and divalent cation channels. So far, most studies on plant CNGCs have been conducted on heterologous systems. In planta, reverse genetic studies revealed the role of different CNGCs in cation uptake, transport and homeostasis. However, there is little information available about the functional characteristics of plant CNGCs. Among the 20 members of this protein family in Arabidopsis, only AtCNGC2 has been functionally identified as an ion channel; therefore, more functional characterization needs to be done on other members of this protein family. Several CNGCs were suggested to be involved in K+, Ca2+ and Na+ uptake and transport, but available information is scarce. This study investigated the relationship between CNGC10 and ion transport in Arabidopsis, with a particular emphasis on the involvement of CNGC10 in salt tolerance. Arabidopsis thaliana wild type (WT) and two AtCNGC10 antisense lines (A2 and A3) were used to characterise the impact of different level of salt stress on (i) root growth, ion concentration in tissues, ion fluxes across the root surface and intracellular ion concentration and pH at the seedling stage, and (ii) photosynthesis and ion concentration in tissues at the flowering stage. Plants of both antisense lines had higher K+ and lower Ca2+ and Mg2+ concentrations in shoots than WT plants when grown in non-salt control 1/4 Hoagland solution. Altered K+, Ca2+ and Mg2+ internal concentrations in AtCNGC10 antisense lines compared with WT plants under non-salt conditions indicated disturbed long distance ion transport, especially xylem loading/retrieval and/or phloem loading. The results of ion fluxes across the root surface also suggested that AtCNGC10 might be involved in transport of K+, Ca2+ and Mg2+ in tissue. Under sudden salt exposure, higher Na+ efflux and smaller K+ efflux in both antisense lines suggested that AtCNGC10 channels are involved in Na+ and K+ transport. The shoots of AtCNGC10 antisense lines A2 and A3 contained higher Na+ concentrations and significantly higher Na+/K+ ratios compared to WT, resulting in impaired photosynthesis and increased salt sensitivity in A2 and A3 than in WT plants. In contrast, seedlings of both antisense lines exposed to salt stress had lower shoot Na+/K+ ratios and longer roots than WT seedlings, indicating that A2 and A3 were more salt-tolerant than WT in the seedling stage, likely because growth is less dependent on photosynthesis in the seedling than in the flowering stage. These results suggested CNGC gene might play a different role during different developmental stages and in various plant organs.

Made available in DSpace on 2014-08-20T13:59:08Z (GMT). No. of bitstreams: 1 tese_maria_da_graca_de_souza_lima.pdf: 4326568 bytes, checksum: 1e4f3676ef163392730daec1cb217796 (MD5) Previous issue date: 2008-07-18 / In Rio Grande do Sul State, the main system for irrigation of rice cultivation is by flooding, can lead to the salinization of soils with inadequate drainage, especially at the coastal region where crops using water from the Laguna dos Patos, which is subject to the salinization by sea water. This is a major environmental problem in the rice production. This survey aimed to examine the expression of enzyme in Oryza sativa L. ssp. indica S. Kato e Oryza sativa L. ssp. japonica S. Kato, grown in different levels of salinity, in order to identify genes involved in tolerance to salinity, based on the assumption that the second subspecies show greater tolerance to salinity. In the experiment were used Oryza sativa L. ssp. japonica S. Kato (BRS Bojuru, IAS 12-9 Formosa and Goyakuman) and Oryza sativa L. ssp. indica S. Kato (BRS-7 Taim, BRS Querência and BRS Atalanta). The seedling was done in plastic trays, containing sand washed as substrate. The seedlings were transferred to greenhouse with 10 days of emergency under temperature 25 °C and humidity 85 % controlled and placed in basins of 15 L containing nutrient solution of Hoagland half strength increased of 0, 25, 50, 75 and 100 mM NaCl. Seedlings were collected at 14, 28, 42 and 56 days after the transfer and immediately stored in ultrafreezer to -70 °C to subsequent analyses. The plant tissues were macerated and placed in tubes eppendorf with extractor solution of Scandálios. The electrophoresis was performed in 7% of polyacrylamide gels placed in vertical vats. The bands were revealed for several enzymes systems: superoxide dismutase, peroxidase, catalase, esterase, xvi glutamate dehydrogenase, alcohol dehydrogenase, fosfoglucose isomerase, malate dehydrogenase, málica enzyme, alpha amylase and glucose-6-phosphate dehydrogenase. Through the search in silico, conducted with the National Center for Biotechnology Information identified the genes AY785147 - SOS and AF319481 - CK1 involved in the salinity tolerance. The detection of the gene was the extraction of DNA using the method CTAB 2%, followed by reactions of PCR thermocycler held on through the use of primers also drawn in silico. The products of amplification were detected by agarose gel electrophoresis of 1.5%. The view of the DNA stained with bromide etídio was made on ultraviolet light and scanned images. The expression of enzymes involved in the mechanisms of tolerance to salt stress is greater in O. sativa ssp. japonica. Fragment of SOS1 gene was found in all cultivars, except for BRS Atalanta. CK1 gene is present in all cultivars evaluated. It allows to conclude that enzyme systems were more expressed in cultivars O. sativa ssp. japonica, in the leaves and the 14 DAT, featuring bands more intense as the increase of salinity. The expression of enzymes involved in the mechanisms of tolerance to salt stress is greater in O. sativa ssp. japonica and the genes studied are present in both subspecies. / No Rio Grande do Sul, o principal sistema de irrigação da cultura do arroz é por inundação, podendo conduzir à salinização os solos com drenagem inadequada, especialmente as lavouras da região litorânea que utilizam a água da Laguna dos Patos, que está sujeita à salinização pela entrada do mar quando é baixo o nível da referida Laguna, tornando-se uma das maiores limitações ambientais na produção de arroz. Esta pesquisa teve como objetivos analisar a expressão enzimática de cultivares de Oryza sativa L. ssp. indica S. Kato e Oryza sativa L. ssp. japonica S. Kato, crescidas em diferentes níveis de salinidade e detectar genes envolvidos com a tolerância à salinidade, com base na hipótese de que as cultivares da Segunda subespécie apresentam maior tolerância à salinidade. No experimento foram utilizadas as cultivares BRS Bojuru, IAS 12-9 Formosa e Goyakuman pertencentes à O. sativa ssp. japonica e as cultivares de O. sativa ssp. indica BRS-7 Taim, BRS Querência e BRS Atalanta. As plântulas de arroz com 10 dias após a emergência (DAE) foram transferidas para casa de vegetação com temperatura e umidade controlada e crescidas em bacias de 15 L, contendo solução nutritiva de Hoagland meia força acrescida de 0, 25, 50, 75 e 100 mM de NaCl. As plântulas foram coletadas aos 14, 28, 42 e 56 dias após a transferência (DAT) e imediatamente armazenadas em ultrafreezer à -70 °C para posterior anáxiv -lises. Os tecidos vegetais foram macerados e colocados em tubos eppendorf com solução extratora de Scandálios. A eletroforese foi realizada em géis de poliacrilamida 7% colocados em cubas eletroforéticas verticais. As bandas foram reveladas para os sistemas enzimáticos superóxido dismutase, peroxidase, catalase, esterase, glutamato desidrogenase, álcool desidrogenase, fosfoglucose isomerase, malato desidrogenase, enzima málica, alfa amilase e glucose-6-fosfato desidrogenase. Por intermédio de pesquisa in silico, realizada junto ao National Center for Biotechnology Information foram identificados os genes AY785147 SOS e AF319481 - CK1, envolvidos na tolerância a salinidade. A detecção dos genes consistiu da extração de DNA genômico segundo o método CTAB 2%, seguido de reações de PCR realizadas em termociclador mediante a utilização dos primers desenhados também in silico. Os produtos da amplificação foram detectados por eletroforese em gel de agarose 1,5%. A visualização do DNA corado com brometo de etídio foi feita sobre iluminação ultravioleta e as imagens digitalizadas. A expressão das enzimas envolvidas nos mecanismos de tolerância ao estresse salino é maior em O. sativa ssp. japonica. Fragmento do gene SOS1 foi encontrado em todas cultivares, com exceção da BRS Atalanta e o gene CK1 está presente em todas as cultivares avaliadas. Conclui-se que os sistemas enzimáticos são mais expressos nas cultivares de O. sativa ssp. japonica, nas folhas e aos 14 DAT, apresentando bandas mais intensas conforme o aumento da salinidade. A expressão das enzimas envolvidas nos mecanismos de tolerância ao estresse salino é maior em O. sativa ssp. japonica e os genes estudados estão presentes nas duas subespécies.

The objective of this project is to develop an operational scheduling model for Sasol Mining’s coal handling facility, Sasol Coal Supply (referred to as SCS), to optimise daily operations. In this document, the specific scheduling problem at SCS is presented and solved using Mixed Integer Non-Linear Programming (MINLP) continuous time representation techniques. The most recent MINLP scheduling techniques are presented and applied to an example problem. The assumption is made that the results from the example problem will display trends which will apply to the SCS scheduling problem as well. Based on this assumption, the unit-specific event based continuous time formulation is chosen to apply to the SCS scheduling problem. The detail mathematical formulation of the SCS scheduling problem, based on the chosen technique, is discussed and the necessary changes presented to customise the formulation for the SCS situation. The results presented show that the first phase model does not solve within 72 hours. A solution time of more than three days is not acceptable for an operational scheduling model in a dynamic system like SCS. Various improvement approaches are applied during the second phase of the model development. Special Ordered Sets of Type 1 (SOS1) variables are successfully applied in the model to reduce the amount of binary variables. The time and duration constraints are restructured to simplify the structure of the model. A specific linearization and solution technique is applied to the non-linear equations to ensure reduced model solution times and reliable results. The improved model for one period solves to optimality within two minutes. This dramatic improvement ensures that the model will be used operationally at SCS to optimise daily operations. The scheduling model is currently being implemented at SCS. Examples of the input variables and output results are presented. It is concluded that the unit-specific event based MINLP continuous time formulation method, as presented in the literature, is not robust enough to be applied to an operational industrial-sized scheduling problem such as the SCS problem. Customised modifications to the formulation are necessary to ensure that the model solves in a time acceptable for operational use. However, it is proved that Mixed Integer Non-linear Programming (MINLP) can successfully be applied to optimise the scheduling of an industrial-sized plant such as SCS. Although more research is required to derive robust formulation techniques, the principle of using mathematical methods to optimise operational scheduling in industry can dramatically impact the way plants are operated. The optimisation of daily schedules at SCS by applying the MINLP continuous time scheduling technique, has made a significant contribution to the coal handling industry. Finally, it can be concluded that the SCS scheduling problem was successfully modelled and the operational scheduling model will add significant value to the Sasol Group. / Dissertation (MEng (Industrial Engineering))--University of Pretoria, 2006. / Industrial and Systems Engineering / unrestricted