Joaquim Enéas-Filho

Effects of salt stress on plant growth, stomatal response and solute accumulation of different maize genotypes

Seeds from eight different maize genotypes (BR3123, BR5004, BR5011, BR5026, BR5033, CMS50, D766 and ICI8447) were sown in vermiculite, and after germination they were transplanted into nutrient solution or nutrient solution containing 100 mmol.L-1 of NaCl and placed in a greenhouse. During the experimental period plant growth (dry matter, shoot to root dry mass ratio, leaf area, relative growth rate, and net assimilation rate), leaf temperature, stomatal conductance, transpiration, predawn water potential, sodium, potassium, soluble amino acids and soluble carbohydrate contents were determined in both control and salt stressed plants of all genotypes studied. Salt stress reduced plant growth of all genotypes but the genotypes BR5033 and BR5011 were characterized as the most salt-tolerant and salt-sensitive, respectively. Stomatal response of the salt-tolerant genotype was not affected by salinity. Among the studied parameters, shoot to root dry mass ratio, leaf sodium content and leaf soluble organic solute content showed no relation with salt tolerance, i.e., they could not be considered as good morpho-physiological markers for maize salt tolerance. In contrast, sodium and soluble organic solutes accumulation in the roots as a result of salt stress appeared to play an important role in the acclimation to salt stress of the maize genotypes studied, suggesting that they could be used as physiological markers during the screening for salt tolerance.

Growth and Protein Pattern in Cowpea Seedlings Subjected to Salinity

Cowpea (Vigna unguiculata) seeds were put to germinate on filter paper under control (distilled water) and salt stress (100 mM MaCl) conditions. Seeds and seedlings were classified in eight developmental stages (DS), according to their morphological traits. Under control conditions, 7 d after planting, 100 % of the seedlings reached DS VIII (seedlings with radicles measuring more than 5 cm, cotyledons leaving the filter paper, hypocotyls straight and cotyledonary leaves fully open) and under NaCl stress conditions, 11 d after planting only 68 % of the seedlings were at DS VIII. The length of the main root and of shoot has decreased 23 and 44 %, respectively. The two-dimensional electrophoretic patterns of the albumins isolated from stems and leaves were determined in seedlings at DS VIII. In stems 19 proteins (14.6 to 76.3 kDa) had their relative concentration increased by salinity, 8 (31.2 to 65.0 kDa) had their relative concentration decreased by salinity and 9 (16.3 to 39.8 kDa) were apparently synthesised de novo. In leaves, under salt conditions 9 proteins (18.2 to 33.2 kDa) increased in concentration, one (17.1 kDa) decreased in concentration and one (21.2 kDa) was apparently synthesised de novo.

Silicon application on plants of maize and cowpea under salt stress

Although silicon is not considered an essential nutrient for plants, it can increase the yield potential of some crops and has been used to mitigate the toxic effects of salt stress. The aim of this work was to evaluate the effect of sodium silicate applied in two different ways in maize and cowpea seedlings subjected to salinity. Seedlings were grown in Hoagland solutions and maintained in a greenhouse. Seedlings received 1.0 mM Na 2 SiO 3 applied directly in the nutrient solutions or by foliar supply, and they were subjected to 100 mM NaCl for 15 days. We evaluated the dry weight of leaves, stems and roots, leaf area and ion leakage in leaves and roots. Salinity reduced dry weight of leaves, stems and roots and increased leaves and root ion leakage. Silicon application in the nutrient solution significantly increased all growth parameters and decreased ion leakage in maize seedlings, whereas this response was not observed in cowpea. Silicon was able to relieve the toxic effects of NaCl on growth only in maize seedlings when applied directly in the nutrient solution.

Purification and Properties of a Ribonuclease from Cowpea Cotyledons

The isolation and characterisation of cotyledonary ribonucleases (RNase; EC 3.1.27.1), are basic steps to understand the physiology and biochemistry of RNA turnover and mobilisation during seed germination and seedling establishment, as well as how environmental stresses affect them. RNase was isolated and purified 928-fold, to apparent electrophoretic homogeneity from 5-d-old seedlings of Vigna unguiculata. It is a protein with an apparent molecular mass of 16 kDa having three major isoforms. Its optimum pH is 5.8, which decreases to 5.2 in presence of KCl. It has an apparent Km of 0.80 mg RNA cm-3 and retains 40 % of its activity when heated to 80 °C. It is completely inhibited by Cu2+, Hg2+ and Zn2+ and is almost insensitive to Mg2+, Ca2+- and EDTA. Urea, Fe2+, Co2+ and 2-mercaptoethanol partially inhibit its activity. Its amino acid composition shows a resem lance to that of other plant RNases.

Multiple forms of cotyledonary b-galactosidases from Vigna unguiculata quiescent seeds

Cotyledonary b-galactosidases were isolated and partially purified from Pitiuba 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 Pitiuba cowpea quiescent seeds possess four isoforms of b-galactosidases.

O estresse salino retarda o desenvolvimento morfofisiológico e a ativação de galactosidases de parede celular em caules de Vigna unguiculata

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, Pitiuba 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.

Purification and characterization of cytosolic and cell wall β-galactosidases from Vigna unguiculata stems

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. Pitiuba 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 KM 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.