Deep RNAseq indicates protective mechanisms of cold-tolerant indica rice plants during early vegetative stage.

KEY MESSAGE: Cold-tolerance in rice may be related to increased cellulose deposition in the cell wall, membrane fatty acids unsaturation and differential expression of several newly identified genes. Low temperature exposure during early vegetative stages limits rice plant's growth and development. Most genes previously related to cold tolerance in rice are from the japonica subspecies. To help clarify the mechanisms that regulate cold tolerance in young indica rice plants, comparative transcriptome analysis of 6 h cold-treated (10 °C) leaves from two genotypes, cold-tolerant (CT) and cold-sensitive (CS), was performed. Differentially expressed genes were identified: 831 and 357 sequences more expressed in the tolerant and in the sensitive genotype, respectively. The genes with higher expression in the CT genotype were used in systems biology analyses to identify protein-protein interaction (PPI) networks and nodes (proteins) that are hubs and bottlenecks in the PPI. From the genes more expressed in the tolerant plants, 60% were reported as affected by cold in previous transcriptome experiments and 27% are located within QTLs related to cold tolerance during the vegetative stage. Novel cold-responsive genes were identified. Quantitative RT-PCR confirmed the high-quality of RNAseq libraries. Several genes related to cell wall assembly or reinforcement are cold-induced or constitutively highly expressed in the tolerant genotype. Cold-tolerant plants have increased cellulose deposition under cold. Genes related to lipid metabolism are more expressed in the tolerant genotype, which has higher membrane fatty acids unsaturation, with increasing levels of linoleic acid under cold. The CT genotype seems to have higher photosynthetic efficiency and antioxidant capacity, as well as more effective ethylene, Ca2+ and hormone signaling than the CS. These genes could be useful in future biotechnological approaches aiming to increase cold tolerance in rice.

Nutritional disorder in Pfaffia glomerata by mercury excess in nutrient solution / Desordem nutricional em Pfaffia glomerata pelo excesso de mercúrio em solução nutritiva

ABSTRACT: The mineral nutritional homeostasis in response to different concentrations of Hg (0, 25 and 50μM) was evaluated in Pfaffia glomerata plant. The exposure to the highest level of Hg (50µM) caused a decreasing in shoot and root fresh weights of 15.5% and 20%, respectively. Both shoot and root Hg concentrations increased linearly with increasing external Hg concentrations. Ca concentration decreased in shoot only at 50µM Hg, whereas shoot K and Mg concentrations decreased at both 25 and 50µM Hg, when compared to the control. A significant decrease in Cu, Zn, Fe and Mn concentrations in plants exposed to Hg was observed, but most Zn, Mn, and Cu in the roots. On the other hand, P concentration increased in both root and shoot of plants exposed at 25 and 50µM Hg, whereas Na concentration increased only in the root at 25 and 50µM Hg exposure. In general, tissue nutrient concentrations in P. glomerata plantlets exposed to Hg were significantly decreased, which indicates that the Hg may cause alteration on the mineral nutritional homeostasis of this species.

RESUMO: A homeostase nutricional mineral em resposta a diferentes concentrações de Hg (0, 25 e 50μM) foram avaliadas em plantas de Pfaffia glomerata. A exposição ao mais alto nível de Hg (50µM) causou um decréscimo de 15,5% e 20%, respectivamente, na matéria fresca da parte aérea e raízes. As concentrações de Hg na parte aérea e raízes aumentaram linearmente com o aumento das concentrações de Hg. A concentração de Ca decresceu na parte aérea somente em 50µM Hg, enquanto as concentrações de K e Mg na parte aérea decresceram tanto em 25 como em 50µM Hg, quando comparado ao controle. Observou-se um significativo decréscimo nas concentrações de Cu, Zn, Fe e Mn nas plantas expostas ao Hg, mas principalmente Zn, Mn e Cu nas raízes. Por outro lado, a concentração de P aumentou em raízes e parte aérea de plantas expostas a 25 e 50µM Hg, enquanto a concentração de Na aumentou somente nas raízes em 25 e 50µM Hg. No geral, as concentrações de nutrientes nos tecidos de P. glomerata expostas ao Hg foram significativamente diminuídas, o que indica que o Hg pode causar alterações na homeostase nutricional mineral dessa espécie.

Cold tolerance in rice germinating seeds revealed by deep RNAseq analysis of contrasting indica genotypes.

Rice productivity is largely affected by low temperature, which can be harmful throughout plant development, from germination to grain filling. Germination of indica rice cultivars under cold is slow and not uniform, resulting in irregular emergence and small plant population. To identify and characterize novel genes involved in cold tolerance during the germination stage, two indica rice genotypes (sister lines previously identified as cold-tolerant and cold-sensitive) were used in parallel transcriptomic analysis (RNAseq) under cold treatment (seeds germinating at 13 °C for 7 days). We detected 1,361 differentially expressed transcripts. Differences in gene expression found by RNAseq were confirmed for 11 selected genes using RT-qPCR. Biological processes enhanced in the cold-tolerant seedlings include: cell division and expansion (confirmed by anatomical sections of germinating seeds), cell wall integrity and extensibility, water uptake and membrane transport capacity, sucrose synthesis, generation of simple sugars, unsaturation of membrane fatty acids, wax biosynthesis, antioxidant capacity (confirmed by histochemical staining of H2O2), and hormone and Ca(2+)-signaling. The cold-sensitive seedlings respond to low temperature stress increasing synthesis of HSPs and dehydrins, along with enhanced ubiquitin/proteasome protein degradation pathway and polyamine biosynthesis. Our findings can be useful in future biotechnological approaches aiming to cold tolerance in indica rice.

Aluminum accumulation in two Pfaffia glomerata genotypes and its growth effects / Acumulação de alumínio em dois genótipos de Pfaffia glomerata e seus efeitos no crescimento

Aluminum (Al) toxicity is a limiting factor for crop production in acid soils, which cover approximately 60% of the Brazilian territory. This study aimed to evaluate the effects of Al on growth and tissue Al concentration of two Pfaffia glomerata accessions (BRA and JB/UFSM). Plantlets were grown in a hydroponic system with five Al concentrations (0, 50, 100, 150 and 200mg L-1) for 7 days. Most of the evaluated parameters presented significant interaction between both P. glomerata accessions and Al levels in nutrient solution and, in general, Al treatments negatively affected plant growth, especially roots. Moreover, BRA accession showed higher Al accumulation in its tissues than JB/UFSM and, consequently in BRA accession the growth was impaired substantially. Furthermore, the results suggest that, between P. glomerata accessions studied, BRA is less appropriated for medicinal uses when grown in soils with high Al levels, due to the higher accumulation of tissue Al content. .

A toxidez do alumínio (Al) é um fator limitante da produção agrícola em solos ácidos, os quais cobrem cerca de 60% do território Brasileiro. O objetivo deste estudo foi avaliar os efeitos do Al no crescimento e na concentração de Al nos tecidos de dois acessos de Pfaffia glomerata (BRA e JB/UFSM). As plantas foram cultivadas em sistema hidropônico, contendo cinco concentrações de Al (0, 50, 100, 150 e 200mg L-1) por 7 dias. Para a maioria dos parâmetros avaliados, houve interação significativa entre os dois acessos de P. glomerata e as concentrações de Al, sendo que, de modo geral, os tratamentos com Al afetaram negativamente o crescimento das plantas, especialmente as raízes. Além disso, o acesso BRA acumulou mais Al nos tecidos que o acesso JB/UFSM e, consequentemente, os parâmetros de crescimento foram afetados mais significativamente naquele acesso. Portanto, nossos resultados sugerem que, entre os acessos de P. glomerata estudados, BRA é menos indicado para usos medicinais quando cultivado em solos com altos níveis de Al, por acumular mais Al em seus tecidos.

Differential speed of activation in antioxidant system in three oat genotypes.

The objective of this study was to evaluate whether the oxidative stress caused by aluminum (Al) toxicity is a symptom that can trigger root growth inhibition in oat genotype seedlings. Oat seedlings were grown in a nutrient solution (pH 4.0) with 0 and 370 µM Al. At 12, 24, and 36 h after Al addition, growth (root length) and biochemical parameters (catalase - CAT, ascorbate peroxidase - APX, and superoxide dismutase - SOD activities, lipid peroxidation, ascorbic acid (ASA) and non-protein thiol group (NPSH) concentration) were determined. The aluminum content was measured in oat seedlings. Regardless of the exposure time, root of the tolerant genotype grew normally with any Al treatments. Al supply caused lipid peroxidation only in the Al-sensitive genotype in roots and shoots (at 12, 24, and 36 h). In sensitive genotype seedlings, CAT, APX, and SOD were activated only at 24 or 36 h. In tolerant and intermediate genotypes, CAT, APX, and SOD were activated at 12, 24, and 36 h. Data for root growth and lipid peroxidation suggested that lipid peroxidation in the sensitive genotype may be an effect of Al toxicity on root growth. Therefore, the tolerant, intermediate, and sensitive genotypes differ in the expression of the amount, type of antioxidants, and speed of activation of antioxidant system, suggesting a varying capacity of these genotypes to deal with oxidative stress, which resulted in varying sensitivity and tolerance to Al.

Zinc alleviates mercury-induced oxidative stress in Pfaffia glomerata (Spreng.) Pedersen.

The possible role of zinc (Zn) to reverse the oxidative stress caused by mercury (Hg) was investigated in Pfaffia glomerata plantlets. Thirty-day-old acclimatized plantlets of P. glomerata were exposed to four treatments: control, 50 µM Zn, 50 µM Hg and 50 µM Zn + 50 µM Hg for 9 days. In Zn + Hg treatment, shoot and root Hg concentrations were 59 and 24% smaller than that plants exposed to 50 µM Hg added alone. An increase in the Zn concentration in the shoot of plants exposed to Zn + Hg occurred, although in the roots Zn concentration was not altered, when compared to the control. Fresh and dry weights, as well as the activity of δ-aminolevulinic acid dehydratase (δ-ALA-D) in Hg-treated plants were significantly reduced. Percentage survival, fresh and dry weights and δ-ALA-D activity of plants treated by 50 µM Zn + 50 µM Hg were greater than of that treated by Hg alone. Moreover, Zn treatment reduced the lipid peroxidation caused by Hg, being this effect related to increased root superoxide dismutase activity, and shoot catalase and ascorbate peroxidase activities. In conclusion, the presence of Zn in the substrate caused a significant reduction in the oxidative stress induced by Hg.

Differential responses of oat genotypes: oxidative stress provoked by aluminum.

The phytotoxic effects of aluminum and the mechanisms of genetically-based Al tolerance have been widely investigated, as reported in many papers and reviews. However, investigations on many Al-sensitive and Al-resistant species demonstrate that Al phytotoxicity and Al-resistance mechanisms are extremely complex phenomena. The objective of the present study was to analyze the effects of aluminum on the activity of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX). Also was evaluated the lipid peroxidation, H(2)O(2) content, levels of ascorbic acid (ASA), non-protein thiols (NPSH) and Al content in three genotypes of oat, Avena sativa L. (UFRGS 930598, UFRGS 17, and UFRGS 280). The genotypes were grown in different concentrations of Al ranging from 90 to 555 µM for 5 days. The antioxidant system was unable to overcome toxicity resulting in negative effects such as lipid peroxidation and H(2)O(2) content in UFRGS 930598. The results showed that UFRGS 930598 was the most sensitive genotype. UFRGS 17 and UFRGS 280 were more resistant to Al toxicity. These results suggest that UFRGS 17 has mechanisms of external detoxification and UFRGS 280 has mechanisms of internal detoxification. The different behavior of enzymatic and non-enzymatic antioxidants of the genotypes showed that aluminum resistance in UFGRS 17 and UFRGS 280 may be related to oxidative stress.

Aluminum-induced oxidative stress in cucumber.

Aluminum (Al) is one of the most abundant elements of the planet and exposure to this metal can cause oxidative stress and lead to various signs of toxicity in plants. Plants are essential organisms for the environment as well as food for humans and animals. The toxic effect of aluminum is the major cause of decreased crop productivity. Thus, the objective of the present study was to analyze the effects of aluminum on the activity of antioxidant enzymes such as catalase (CAT - E.C. 1.11.1.6), superoxide dismutase (SOD - E.C.1.15.1.1) and ascorbate peroxidase (APX - E.C. 1.11.1.11), and on lipid peroxidation, electrolyte leakage percentage (ELP) and chlorophyll and protein oxidation levels in Cucumis sativus L. (cv. Aodai). Seedlings were grown at different concentrations of aluminum ranging from 1 to 2000 microM for 10 days. The increase in ELP and H(2)O(2) production observed in the seedlings may be related to the decreased efficiency of the antioxidant system at higher aluminum concentrations. The antioxidant system was unable to overcome toxicity resulting in negative effects such as lipid peroxidation, protein oxidation and a decrease in the growth of Cucumis seedlings. Aluminum toxicity triggered alterations in the antioxidant and physiological status of growing cucumber seedlings.

Oxidative stress is an early symptom triggered by aluminum in Al-sensitive potato plantlets.

The objective of this study was to evaluate whether the oxidative stress caused by aluminum (Al) toxicity is an early symptom that can trigger root growth inhibition in Macaca (Al-sensitive) and SMIC148-A (Al-tolerant) potato clones. Plantlets were grown in a nutrient solution (pH 4.00) with 0, 100 and 200mg Al L(-1). At 24, 72, 120 and 168h after Al addition, root length and biochemical parameters were determined. Regardless of exposure time, root length of the Macaca clone was significantly lower at 200mg Al L(-1). For the SMIC148-A clone, root length did not decrease with any Al treatments. Al supply caused lipid peroxidation only in Macaca, in both roots (at 24, 72, 120 and 168h) and shoot (at 120 and 168h). In roots of the Macaca, catalase (CAT) and ascorbate peroxidase (APX) activity decreased at 72 and 120h, and at 24, 72 and 120h, respectively. At 168h, both activities increased upon addition of Al. In roots of the SMIC148-A, CAT activity increased at 72 and 168h, whereas APX activity decreased at 72h and increased at 24, 12 and 168h. The Macaca showed lower root non-protein thiol group (NPSH) concentration at 200mg Al L(-1) in all evaluations, but the SMIC148-A either did not demonstrate any alterations at 24 and 72h or presented higher levels at 120h. This pattern was also observed in root ascorbic acid (AsA) concentration at 24 and 120h. The cellular redox status of these potato clones seems to be affected by Al. Therefore, oxidative stress may be an important mechanism for Al toxicity, mainly in the Al-sensitive Macaca clone.

Micronutrient concentration in potato clones with distinct physiological sensitivity to Al stress / Concentração de micronutrientes em clones de batata com distinta sensibilidade fisiológica ao estresse de alumínio

The objective of this study was to evaluate the effects of aluminum (Al) on the zinc (Zn), manganese (Mn), iron (Fe) and copper (Cu) concentrations in four potato clones (Macaca and Dakota Rose: both Al-sensitive clones; and SMIC148-A and Solanum microdontum: both Al-tolerant-clones), grown in a nutrient solution (pH 4.00) with 0, 50, 100, 150 and 200mg Al L-1. Root Zn and Fe concentrations decreased linearly with the increase of Al levels in Macaca, SMIC148-A and Dakota Rose and increased linearly in S. microdontum. Shoot Zn concentration showed a quadratic relationship with Al in S. microdontum and SMIC148-A, but a curvilinear response in Dakota Rose. Shoot Fe concentration showed a quadratic relationship with Al in S. microdontum, SMIC148-A and Dakota Rose. Root Mn concentration decreased linearly in Macaca and SMIC148-A, and increased linearly in S. microdontum with Al levels. Mn concentration showed a quadratic relationship with Al in roots of Dakota Rose and in shoot of SMIC148-A, and increased curvilinearly with Al levels in shoot of Dakota Rose. In shoot, there was no alteration in Zn, Fe and Mn in Macaca and Mn concentration in S. microdontum. Roots and shoot Cu concentration increased linearly in Dakota Rose, and showed quadratic relationship with Al in Macaca. Roots Cu concentration showed a quadratic relationship with Al levels in S. microdontum and SMIC148-A. Shoot Cu concentration increased linearly in S. microdontum, and decreased linearly in SMIC148-A. Therefore, the excessive Al accumulation affected the uptake and distribution of Zn, Fe, Mn and Cu in roots and shoot of potato clones.The response of shoot Cu concentration to Al was less altered in the Al-tolerant clones than was in Al-sensitive clones. Aluminum tolerance in S. microdontum may be connected with greater levels of Zn, Fe and Mn in the roots.

O objetivo deste estudo foi caracterizar o efeito do alumínio (Al) na concentração de zinco (Zn), manganês (Mn), ferro (Fe) e cobre (Cu) em quatro clones de batata (Macaca e Dakota Rose: sensíveis ao Al; e SMIC148-A e Solanum microdontum: tolerantes ao Al) crescendo em solução nutritiva (pH 4,0) com 0, 50, 100, 150 e 200mg Al L-1. A concentração de Zn e Fe em raízes diminuiu linearmente com o aumento dos níveis de Al nos clones Macaca, SMIC148-A e Dakota Rose e aumentou linearmente em S. microdontum. Na parte aérea, a concentração de Zn mostrou resposta quadrática ao Al em S. microdontum e SMIC148-A, enquanto no clone Dakota Rose houve uma resposta cúbica. Nos clones S. microdontum, SMIC148-A e Dakota Rose, a concentração de Fe mostrou resposta quadrática ao Al. A concentração de Mn em raízes diminuiu linearmente em relação ao Al nos clones Macaca e SMIC148-A e aumentou linearmente em S. microdontum. Para Dakota Rose e SMIC148-A, a concentração de Mn mostrou uma resposta quadrática em relação ao Al em raízes e parte aérea. A concentração de Mn na parte aérea aumentou de forma cúbica com os níveis de Al no clone Dakota Rose. Na parte aérea, não houve alteração na concentração de Zn e Fe na Macaca e de Mn nos clones Macaca e S. microdontum. Em raízes e na parte aérea, a concentração de Cu aumentou linearmente no clone Dakota Rose e mostrou resposta quadrática no clone Macaca. A concentração de Cu mostrou resposta quadrática com os níveis de Al em raízes dos clones S. microdontum e SMIC148-A. Na parte aérea, a concentração de Cu aumentou linearmente no clone S. microdontum e diminuiu linearmente no clone SMIC148-A com o aumento nos níveis de Al. Portanto, a acumulação excessiva de Al afetou negativamente a absorção e a distribuição de Zn, Fe, Mn e Cu nas raízes e na parte aérea dos clones de batata. A resposta da concentração de Cu na parte aérea ao Al foi menos alterada nos clones Al-tolerantes que naqueles Al-sensíveis. A tolerância ao...

Presence of multiple acid phosphatases activity in seedlings of cucumber, radish and rocket salad

Acid phosphatases (3.1.3.2) are a group of enzymes widely distributed in nature, which catalyze the hydrolysis of a variety of phosphate esters in the pH range of 4-6. We confirmed the presence of acid phosphatases in seedlings of cucumber (Cucumis sativus), radish (Raphanus sativus) and rocket salad (Eruca vesicaria) under different assay conditions using a rapid and simple preparation. The results showed that the optimum pH and temperature used for all species were close to 5.5 and 35°C, respectively. The enzyme was inhibited by molybdate, fluoride, azide, levamisole, orthovanadate, Zn2+ and Cu2+. Suramin had no effect on enzyme activity. The acid phosphatase from cucumber, radish and rocket salad hydrolyzed a wide variety of phosphate esters and the highest activity was observed with PPi, ATP and GTP. These results demonstrate that the enzyme investigated in this study is different from well known ester phosphate cleaving plant enzymes (apyrase and inorganic pyrophosphatases) and this preparation could be a useful tool to future toxicological studies and to study initially all isoforms of acid phosphatase.

As fosfatases ácidas (3.1.3.2) são um grupo de enzimas amplamente distribuídas na natureza, as quais catalisam a hidrólise de uma variedade de ésteres de fosfato com uma variação de pH entre quatro e seis. Foi confirmada a presença de fosfatases ácidas em plântulas de pepino (Cucumis sativus), rabanete (Raphanus sativus) e rúcula (Eruca vesicaria) sob diferentes condições de ensaio usando uma preparação rápida e simples. Os resultados mostraram que o pH e a temperatura ótimos para todas as espécies foram 5,5 e 35°C, respectivamente. A enzima foi inibida por molibdato, fluoreto, azida, levamisole, ortovanadato, Zn2+ e Cu2+. O inibidor suramim não afetou a atividade enzimática. As fosfatases ácidas de pepino, rabanete e rúcula hidrolisaram uma ampla variedade de ésteres de fosfato e a maior atividade foi observada com PPi, ATP e GTP para pepino e rabanete e PPi, frutose-6-fosfato e GTP para rúcula. Esses resultados demonstraram que a enzima investigada neste estudo é diferente das conhecidas enzimas de plantas que clivam ésteres de fosfato (apirase e pirofosfatases inorgânicas). Desse modo, esta preparação pode ser uma ferramenta útil para futuros estudos toxicológicos e para se estudar inicialmente todas as isoformas das fosfatases ácidas.

Malathion, carbofuran and paraquat inhibit Bungarus sindanus (krait) venom acetylcholinesterase and human serum butyrylcholinesterase in vitro.

Carbofuran and malathion, well known pesticides, and paraquat, a world widely used herbicide, were tested on acetylcholinesterase (AChE) from Bungarus sindanus venom and butyrylcholinesterase (BChE) from human serum. The calculated IC(50 )values for inhibition of venom enzyme by malathion, carbofuran and paraquat were 2.5, 0.14, and 0.16 microM, respectively. The values for inhibition of serum butyrylcholinesterase (BChE) were 3.5, 0.09 and 0.18 microM, respectively. Analysis of kinetic data indicated that the inhibition caused by malathion, carbofuran and paraquat was mixed for venom AChE. For BChE from human serum, the inhibition caused by malathion and paraquat was mixed and for carbofuran it was uncompetitive. The present results suggest a commercial paraquat preparation (a popular herbicide) inhibits cholinesterases with similar or higher potency than classical pesticide inhibitors. Furthermore, this inhibition was observed both in human serum and snake venom, a newly studied source of AChE.

Mercury toxicity induces oxidative stress in growing cucumber seedlings.

In this study, the effects of exogenous mercury (HgCl(2)) on time-dependent changes in the activities of antioxidant enzymes (catalase and ascorbate peroxidase), lipid peroxidation, chlorophyll content and protein oxidation in cucumber seedlings (Cucumis sativus L.) were investigated. Cucumber seedlings were exposed to from 0 to 500microM of HgCl(2) during 10 and 15 days. Hg was readily absorbed by growing seedlings, and its content was greater in the roots than the in shoot. Time and concentration-dependent reduction in root and shoot length was observed at all concentrations tested, equally in the roots and shoot, at both 10 and 15 days. At 50microM HgCl(2), root fresh weight of 15-day-old seedlings increased, and at other concentrations, it reduced. For 10-day-old seedlings, reduction in root and shoot fresh biomass was observed. At 15 days, only at 50microM HgCl(2) was there no observed reduction in shoot fresh biomass. Dry weight of roots increased at 500microM both at 10 and 15 days, though at 250microM HgCl(2) there was only an increase at 15 days. There was a significant effect on shoot dry weight at all concentrations tested. Hg-treated seedlings showed elevated levels of lipid peroxides with a concomitant increase in protein oxidation levels, and decreased chlorophyll content when exposed to between 250 and 500microM of HgCl(2). At 10 days, catalase activity increased in seedlings at a moderately toxic level of Hg, whereas at the higher concentration (500microM), there was a marked inhibition. Taken together, our results suggest that Hg induces oxidative stress in cucumber, resulting in plant injury.

Catalase activity in Candida albicans exposed to antineoplastic drugs.

An increased catalase activity in Candida spp. has been suggested as a mechanism that reduces amphotericin B activity. Furthermore, resistance to antifungal agents like amphotericin B has been reported in some cancer patients undergoing chemotherapy treatment. In this study we analysed the influence of chemotherapy agents on catalase activity in Candida albicans, the major species involved in yeast infections. Eight strains of C. albicans isolated from HIV-positive patients were exposed to cyclophosphamide, cytarabine, dacarbazine and methotrexate antineoplastic drugs at the concentrations used during therapy. Catalase activity was measured and compared to the control group. Very significant differences (P < 0.01) were found when C. albicans was exposed to methotrexate (2 microg ml(-1) = 4 microM). For cyclophosphamide (50 microg ml(-1)), cytarabine (1 microg ml(-1)) and dacarbazine (8 microg ml(-1)), no differences were found (P > 0.05) between the control and drug-exposed groups. Although more extensive studies are necessary, these data do suggest that the antineoplastic drug methotrexate contributes to the resistance to antifungal drug therapy by varying catalase activity.