O objetivo foi avaliar as propriedades e a expressão gênica de isoformas de β-galactosidase isoladas de caules de plântulas de feijão-de-corda cv Pitiúba em diferentes estádios de desenvolvimento semeadas na ausência e presença de NaCl. As sementes foram semeadas em folhas de papel filtro embebidas em água destilada (controle) e em solução de NaCl 100 mM. As plântulas foram coletadas nos estádios de desenvolvimento III (ED III) e VIII (ED VIII). As isoformas da enzima, β-gal I, II e III, apresentaram massas moleculares de 89; 146 e 124 kDa, respectivamente, e o mesmo pH (4,0) e temperatura ótima de ensaio (55 °C), em todas as condições estudadas. As três isoformas mostraram-se estáveis até a temperatura de 50 °C. As curvas de termoinativação em função do tempo para as enzimas mostraram que, na temperatura de 60 °C, as curvas foram bifásicas. As análises da expressão gênica de β-galactosidases foram feitas usando primers degenerados e RNA total de caules de plântulas semeadas em condições controle e com tratamento salino, nos ED III e ED VIII. Os resultados revelaram maior nível de transcritos no ED VIII, com redução dos níveis de transcritos de β-galactosidases quando submetidos ao tratamento salino. Os resultados mostraram que as β-gal I, II e III não sofrem modificações ao longo dos estádios iniciais de desenvolvimento. Há algumas diferenças entre as propriedades das três isoformas. A salinidade afetou a expressão gênica das enzimas. As enzimas foram expressas diferentemente nos ED III e ED VIII.

The aim of this work was to evaluate the properties and gene expression of isoforms of β-galactosidase, isolated from the stems of seedlings of the cowpea cv. Pitiuba, at different stages of development and sown in the absence and presence of NaCl. Seeds were sown onto sheets of filter paper soaked in distilled water (control) and a 100 mM NaCl solution. The plants were collected at the III (ED III) and VIII (ED VIII) stages of development. Isoforms of the enzyme β-gal I, II and III presented molecular weights of 89, 146 and 124 kDa respectively, and the same pH (4.0) and optimum test temperature (55 °C) under all the conditions studied. The three isoforms were stable up to 50 °C. The thermal inactivation curves of the enzymes as a function of time were biphasic at a temperature of 60 °C. Analysis of the gene expression of β-galactosidase was carried out using degenerate primers and total RNA from the stems of seedlings at ED III and ED VIII, sown both as control and under saline treatments. The results showed higher transcript levels at ED VIII, with reduced levels of β-galactosidase transcripts under saline treatment, and that β-gal I, II and III do not undergo changes during the early stages of development. There are a few differences in the properties of the three isoforms. Salinity affected the gene expression of enzymes. The enzymes were expressed differently for ED III and ED VIII.

Com o objetivo de examinar o envolvimento das α- e β-galactosidases na expansão celular de caules de plântulas de feijão-de-corda submetidas a estresse salino durante o estabelecimento da plântula, e de analisar os efeitos do estresse salino no desenvolvimento das plântulas e nas atividades enzimáticas, sementes de feijão-de-corda Pitiúba foram semeadas em água destilada e em solução de NaCl 100 mM. Foram coletados caules em diferentes estádios de desenvolvimento e com diferentes tempos após a semeadura. Avaliou-se o crescimento através das medidas de comprimento e das matérias fresca e seca dos caules. A salinidade tanto inibiu como retardou o crescimento dos caules. Os efeitos do NaCl nas atividades galactosidásicas de parede celular foram estudados in vivo e in vitro. A inibição e o retardamento do crescimento dos caules correlacionaram-se com as variações em atividades galactosidásicas. As galactosidases de parede celular de caules de plântulas tiveram suas atividades inibidas com o aumento da concentração de sal no meio de reação. A partir de 250 mM de NaCl as β-galactosidases foram mais sensíveis ao sal que α-galactosidases.

In order to examine the participation of α- and β-galactosidases in the cell expansion of stems from cowpea seedlings submitted to salt stress during plant establishment as well as to analyze the effect of salt stress on the development of seedlings and enzymatic activities, Pitiúba cowpea seeds were sown in distilled water and in 100 mM NaCl. Throughout seed germination and seedling development, stems were harvested from seedlings at different stages of development and at different times after planting. Growth was evaluated by measuring stem length and fresh and dry mass of stems. Salinity both inhibited and delayed the growth of seedling stems. The effects of NaCl on galactosidase activities of the cell wall were studied, both in vivo and in vitro. Galactosidase activities in vivo were related to effects of NaCl inhibition and delay of stem development. The increase in salt concentration inhibited isolated galactosidase activity of cell wall from stems of seedlings. Starting at 250 mM NaCl, β-galactosidases were more sensitive to salt than α-galactosidases.

Three β-galactosidase isoforms, β-gal I and β-gal II (cytosolic) and β-gal III (cell wall-associated), were isolated from stems of Vigna unguiculata (L.) Walp. cv. Pitiúba seedlings. Purification consisted of aμMonia sulfate fractionation followed by chromatography in DEAE-Sephadex and Lactosyl-Sepharose columns. The two cytosolic isoforms showed the same chromatography pattern, which differed from that of β-gal III. Electrophoresis revealed a single band of protein for β-gal II and β-gal III which also expressed β-galactosidase activity in gel. The apparent molecular mass of the β-gal I, II and III was 89, 146 and 124 kDa, respectively. The three isoforms revealed the same optimal pH (4.0) and the same optimal assay temperature (55ºC) for enzyme activity. The three isoforms were stable at temperatures up to 50ºC, and incubation with glucose and galactose expanded their thermal stability as well as inhibited their activities. Galactose was the most effective in promoting these effects and β-gal I and II were competitively inhibited by this sugar. Kinetic analysis using β-PNPG as substrate, revealed K M of 1.69, 1.76 and 1.43 for β-gal I, β-gal II and β-gal III, respectively. The β-gal I was able to hydrolyze all synthetic substrates tested, whereas β-gal II exhibited only β-fucosidase and α-arabinosidase activities, and β-gal III was limited to the α-galactosidase, β-fucosidase and α-arabinosidase activities. These results are consistent with three distinct β-galactosidases exhibiting quite similar kinetic features, but endowed with different functional specificities probably related to their specific roles in the plant cell physiology.

O feijão-de-corda [Vigna unguiculata (L.) Walp] cv. Pitiúba é uma importante cultura nos âmbitos econômico e social do Nordeste do Brasil, especialmente no Estado do Ceará, que é considerado o maior produtor desta região. Com a finalidade de caracterizar os sintomas de deficiências nutricionais em plântulas de feijão-de-corda, as sementes foram semeadas em areia exaustivamente lavada e, após um período de cinco dias, as plântulas foram transferidas para uma solução nutritiva completa para o período de aclimatação. Após três dias, as plântulas foram submetidas aos diferentes tratamentos. O experimento teve os seguintes tratamentos: solução nutritiva completa (N; P; K; Ca; Mg; S e micronutrientes) e omissão individual de cada elemento -N; -P; -K; -Ca; -Mg; -S; -B; -Fe, bem como ausência de aeração. Os sintomas das deficiências foram observados, caracterizados e registrados por fotografias. No final do experimento, as medidas de comprimento e os teores de matérias fresca e seca das raízes e parte aérea das plântulas foram analisados. Todos os macros e micronutrientes causaram sintomas de deficiência e afetaram o desenvolvimento das plântulas. Os sintomas foram desenvolvidos primeiramente em plântulas com carência em Fe, Ca e N. A matéria seca total foi reduzida em todos os tratamentos com ausência de nutrientes. A ausência de Ca, N e Fe foi responsável por uma maior redução da biomassa. A ordem decrescente de redução foi a seguinte: Ca > N > Fe > P > K > Mg > S > Aeração > B > Completo.

The Cowpea [Vigna unguiculata (L.) Walp] cv. Pitiúba is an important crop in the economic and social contexts in the Northeast of Brazil, especially in the state of Ceará, which is considered the largest producer of this region. With the purpose of characterizing the symptoms of nutritional deficiencies in Cowpea seedlings, the seeds were sowed in washed sand and, after a period of five days, the seedlings were transferred for a complete nutrient solution for the period of acclimatization. After three days, the seedlings were submitted to the different treatments. The experiment had the following treatments: complete nutrient solution (N; P; K; Ca; Mg; S and micronutrients) and individual omission of each element -N; -P; -K; -Ca; -Mg; -S; -B, -Fe and -aeration. The symptoms of the deficiencies were observed, characterized and registered by pictures. In the end of the experiment, the length measures and the fresh and dry matters of the root and shoot of the seedlings were analyzed. All macro and micronutrients caused deficiency symptoms and they affected the development of the seedlings. The symptoms were developed firstly in seedlings with failure in Fe, Ca and N. The total dry matter was reduced in all the treatments with absence of nutrients, but the absence of Ca, N and Fe were responsible for the larger reduction of biomass. The decreasing order of reduction was: Ca > N > Fe > P > K > Mg > S > Aeration > B > Complete.

Três isoformas da enzima beta-galactosidase foram isoladas e purificadas parcialmente a partir de cotilédones de sementes quiescentes dos cultivares Vita 3 e Vita 5 de feijão-de-corda [Vigna unguiculata (L.) Walp.], os quais diferem em tolerância aos estresses hídrico e salino. O processo de purificação consistiu de fracionamento com sulfato de amônio, precipitação ácida, seguida pela associação de cromatografia de troca iônica em coluna de DEAE-Sephadex com cromatografia de afinidade em coluna de Lactosyl-Sepharose. As três isoformas isoladas dos dois cultivares mostraram os mesmos padrões cromatográficos, mesma temperatura ótima de ensaio para atividade enzimática (60ºC), idêntica estabilidade térmica até 50ºC e apresentaram ótimos de pH semelhantes (3-4). Entretanto, diferiram umas das outras em suas sensibilidades a íons metálicos e a certos agentes químicos presentes no meio de reação. Os resultados mostraram que as diferenças observadas nas beta-galactosidases de cotilédones de sementes quiescentes foram insuficientes para correlacioná-las com a tolerância ao estresse.

Three isoforms of beta-galactosidases were isolated and partially purified from the cotyledons of quiescent seeds of Vita 3 and Vita 5 cowpea [Vigna unguiculata (L.) Walp.] cultivars differing in water and salt stress tolerance. The purification procedure consisted of ammonium sulfate fractionation, acid precipitation, ion exchange chromatography through DEAE-sephadex and affinity chromatography through Lactosyl-sepharose columns. The three isoforms isolated from the two cultivars showed the same chromatographic patterns, same optimum of temperature for enzyme activity assay (60ºC), identical thermal stability up to 50°C, and similar pH optima (3-4). However, they differed from each other in sensitivity towards metal ions and certain chemical agents presents in the assay medium. The results have shown that the observed differences in beta-galactosidases from the cotyledons of quiescent seeds were not sufficient to relate them to stress tolerance.

b-galactosidases cotiledonárias foram isoladas e purificadas, parcialmente, de sementes quiescentes de feijão-de-corda (Vigna unguiculata (L.) Walp.) Pitiúba. As etapas de purificação consistiram de precipitação do extrato bruto com sulfato de amônio na faixa de 20-60% de saturação, precipitação ácida, cromatografia de troca-iônica em DEAE-Sephadex e cromatografia de afinidade em Lactosil- Sepharose. Esse processo de purificação deu origem a três frações ricas em b-galactosidases: b-gal I, b-gal II e b-gal III, as quais foram purificadas cerca de 5, 509 e 62 vezes, respectivamente. Elas atingiram máxima atividade enzimática em diferentes faixas de pH: 3,5-4,5 para b-gal I, 3,0-3,5 para b-gal II e 3,0-4,0 para b-gal III. Suas atividades máximas foram alcançadas quando a temperatura do meio de ensaio era 60° C, e a preincubação das enzimas em diferentes temperaturas mostrou que elas eram termoestáveis até 50° C. Não houve diferenças significativas entre as enzimas parcialmente purificadas no que respeita à resposta dos diferentes efetores testados, exceto para Mn2+ e EDTA, que afetaram, diferentemente, b-gal I, b-gal II e b-gal III. Elas foram ligeiramente afetadas por Mg2+, Ca2+, Zn2+, Co2+, tartarato, molibdato, glicose e lactose, fortemente inibidas por Cu2+ e galactose, e inativadas por Hg2+. Essas propriedades químicas e físicas são semelhantes às encontradas para outras b-galactosidases de plantas. Embora, três isoformas dessa enzima tenham sido obtidas através desse processo de purificação, a focalização isoelétrica em placa de gel de poliacrilamida dessas proteinas enzimáticas sugere que cotilédones de sementes quiescentes de feijão-de-corda Pitiúba possuem quatro isoformas de b-galactosidases.

Cotyledonary b-galactosidases were isolated and partially purified from Pitiúba cowpea (Vigna unguiculata (L.) Walp.) quiescent seeds. The purification steps consisted of precipitation of the crude extract with ammonium sulphate in the range of 20-60% saturation, acid precipitation, DEAE-Sephadex ion-exchange chromatography and Lactosyl-Sepharose affinity chromatography. This purification process gave rise to three b-galactosidases-rich fractions: b-gal I, b-gal II and b-gal III, which were purified about 5, 509, and 62 fold, respectively. They reached maximal enzyme activity at different pH ranges: 3.5-4.5 for b-gal I, 3.0-3.5 for b-gal II, and 3.0-4.0 for b-gal III. Their maximal activities were reached when the temperature of the assay medium was 60° C, and preincubation of the enzymes at different temperatures has shown that they were heat-stable up to 50° C. There were no significant differences among the partially purified enzymes as far as their response to the different effectors tested, except for Mn2+ and EDTA, which affected differently b-gal I, b-gal II, and b-gal III. They were slightly affected by Mg2+, Ca2+, Zn2+, Co2+, tartarate, molybdate, glucose, and lactose, strongly inhibited by Cu2+ and galactose, and inactivated by Hg2+. These chemical and physical properties are similar to the ones found for other plant b-galactosidases. Although through this process of purification three isoforms of this enzyme were obtained, isoelectric focusing in polyacrylamide slab gel of these enzyme-proteins suggest that cotyledons of Pitiúba cowpea quiescent seeds possess four isoforms of b-galactosidases.