Dulce De Oliveira

Plant glycine-rich proteins: a family or just proteins with a common motif?

Twelve years ago a set of glycine-rich proteins (GRP) of plants were characterized and since then a wealth of new GRPs have been identified. The highly specific but diverse expression pattern of grp genes, taken together with the distinct sub-cellular localisation of some GRP groups, clearly indicate that these proteins are implicated in several independent physiological processes. Notwithstanding the absence of a clear definition of the role of GRPs in plant cells, studies conducted with these proteins have provided new and interesting insights on the molecular and cell biology of plants. Complex regulated promoters and distinct mechanisms of gene expression regulation have been demonstrated. New protein targeting pathways, as well as the exportation of GRPs from different cell types have been discovered. These data show that GRPs can be useful as markers and/or models to understand distinct aspects of plant biology. In this review, the structural and functional features of this family of plant proteins will be summarised. Special emphasis will be given to the gene expression regulation of GRPs isolated from different plant species, as it can help to unravel their possible biological functions.

Identification of compatible and incompatible interactions between Arabidopsis thaliana and Xanthomonas campestris pv. campestris and characterization of the hypersensitive response

Both compatible and incompatible interactions between Arabidopsis thaliana and Xanthomonas campestris have been identified and, for the first time, a strong hypersensitive response has been characterized. A highly reproducible mass spray inoculation protocol has been established and was used together with the more commonly used infiltration inoculation procedure to study the defense responses occurring in mature A. thaliana plants. A series of bacterial strains have been tested on A. thaliana ecotype Columbia (Col-O). X. c. pv. campestris was the most effective pathogen in these tests and was used for further detailed analysis. Several X. c. pv. campestris isolates were tested on A. thaliana Col-O, and one particular X. c. pv. campestris strain (147) was tested on 27 Arabidopsis ecotypes. Symptom development of compatible and incompatible interactions, including the hypersensitive response, was extensively characterized in A. thaliana Col-O. Lesion structure, bacterial distribution, accumulation of polyphenolic compounds, and the deposit of callose in inoculated leaves were documented by microscopic analysis. Activation of the defense-associated genes coding for phenylalanine ammonia lyase (PAL), beta-1, 3-glucanases, chitinases, and peroxidases was evaluated by Northern blot analysis.

Arabidopsis thaliana class IV chitinase is early induced during the interaction with Xanthomonas campestris

Endochitinases are widely distributed among higher plants, including a number of important crop species. They are generally considered to be involved in plant defence against potential pathogens. We have cloned a class IV chitinase gene (AtchitIV) from Arabidopsis thaliana. Southern blot analysis allowed the detection of two cross‐hybridising genes in the A. thaliana genome. AtchitIV transcripts are detected in seedpods, but not in roots, inflorescence stems, leaves and flowers of healthy plants. The transcripts accumulated very rapidly in leaves after inoculation with Xanthomonas campestris. Maximum mRNA accumulation was reached one hour after infection and decreased to very low levels 72 hours after induction. This result suggests an involvement of AtchitIV in the initial events of the hypersensitive reaction. Nevertheless, A. thaliana plants transformed with the gus gene under the control of a class IV chitinase bean promoter, showed GUS activity in seed embryos. These data, together with the constitutive expression of the endogenous gene in the seedpods, points to additional physiological roles for this protein.

Relationships between trehalose metabolism and maltose utilization in Saccharomyces cerevisiae, II: effect of constitutive MAL genes

SummaryA pattern of active accumulation of trehalose during growth on glucose medium, TAC(+) phenotype, is controlled by a polymeric series of maltose fermentation (MAL) genes. An essential requirement for expression of the TAC(+) phenotype is that the MAL gene be in the constitutive state, MALc. Mutation of a constitutive MAL allele to a maltose- inducible or nonfermenting (mal) state, alters the pattern of trehalose metabolism so that little or no trehalose accumulation occurs during growth on glucose medium. The TAC(+) phenotype is obtained in MALc strains whether or not α-glucosidase formation is sensitive or resistant to carbon catabolite repression. However, trehalose accumulation is sensitive to glucose levels even in MALc strains in which α-glucosidase formation is insensitive to catabolite repression. The effects of constitutive MAL genes on trehalose accumulation cannot be accounted for by an increase in trehalose-6 phosphate synthase or a decrease in trehalase as determined in vitro. A mechanism is proposed in which the gene-product of a MAL gene serves as a common positive regulator for expression of four genes coding respectively for maltose permease, maltase, α-methylglucosidase and a component of the trehalose accumulation system.

Permeabilization of yeast for in situ determination of α-glucosidase

Abstract A quantitative in situ assay of yeast α-glucosidase involving permeabilization of the cells by freezing and thawing is described. The assay was applied to different strains in different physiological states and was shown to give results comparable to those obtained with total cell homogenates. The primary advantage of the in situ assay was the possibility of analyzing a large number of samples from the same culture during a growth curve using a very reduced cell mass.

In vitro regeneration of peanut (Arachis hypogaea L.) through organogenesis : Effect of culture temperature and silver nitrate

SummaryThe effect of culture temperature on the morphogenetic response of Arachis hypogaea was studied. Cotyledons were cultivated on MS medium supplemented with 110 µM 6-benzyladenine. Leaf explants were cultivated in the presence of the same growth regulator at 22 µM. Cultures were incubated at temperatures of 25, 28, and 35±5° C. Both direct organogenesis from cotyledons and development of organogenic calluses from leaves showed optimal rates at 35±5° C. The highest frequency of elongation of buds into shoots from leaf-derived calluses occurred in the presence of 5 µM AgNO3. At the best culture temperature, an average of 95% of shoots formed roots on growth-regulator-free MS medium. Plants were successfully transferred to soil, showing normal phenotypes.

PREFERENTIAL TRANSCRIPTIONAL ACTIVITY OF A GLYCINE-RICH PROTEIN GENE FROM ARABIDOPSIS THALIANA IN PROTODERM-DERIVED CELLS

Glycine-rich proteins (GRPs) are characterized by having the glycine residues arranged in characteristic repetitive structural motifs, but with distinct primary sequence. Although identified in various plant species, little is known about their contribution to the morphology and development of the organism. In this study we have isolated the atgrp-5 gene encoding a glycine-rich protein from Arabidopsis thaliana. The promoter region of atgrp-5 was fused to the gus gene and the tissue expression pattern was analyzed in tobacco and A. thaliana transgenic plants. The chimeric gene displayed similar expression patterns in both species, showing preferential expression in protoderm-derived cells. Activity was also detected in the cortical parenchyma of A. thaliana inflorescence axis and in the stem phloem of tobacco.

Regulation of transformation efficiency of peanut (Arachis hypogaea L.) explants by Agrobacterium tumefaciens

Abstract Some of the factors that modulate the transformation efficiency of regenerable tissues of peanut (Arachis hypogaea L.) were examined, using a tumor induction system based on strain A281 harbouring a binary vector. Factors examined were the composition of media, the cocultivation period, tissue type, bacterial inoculum concentration and pretreatment with acetosyringone (AS). Transformation efficiency was greatest when leaf or cotyledon explants were cocultivated on solid rather than liquid medium for 48 h. Highest transformation rates were observed on leaves from 7–10-day-old seedlings, with inoculum densities of 109-5 × 109 cells/ml. Addition of AS to the bacterial culture prior to inoculation did not alter transformation frequency.

Tissue culture of Hypericum brasiliense Choisy: Shoot multiplication and callus induction

Hypericum brasiliense, a non-domesticated plant has been shown to have useful medicinal properties. This plant has not been cultivated so a protocol for mass propagation based on selection of superior clones was developed and a protocol established for the culture of callus cells that could be used for in vitro metabolite production. A micropropagation method based on amplification of nodal buds was developed, by selection, from ten seedling clones that were examined for growth rate, multiplication rate and rooting. The effect of various basal media, growth regulator types and concentrations were examined for optimal callus induction. Optimal callus induction occured on either Murashige and Skoogs or Gamborgs media supplemented with 1 to 2 mg l−1 of 2,4-dichlorophenoxyacetic acid.

Multiplication of juvenile black wattle by microcuttings

The influence of mineral formulation, growth regulators and activated charcoal on micropropagation of juvenile black wattle (Acacia mearnsii) was studied. Nodal segments of one-month-old seedlings were used as starting material. After 30 to 45 days, they developed into shoots, which were divided into microcuttings and transferred to fresh media. The addition of 2 g l−1 of activated charcoal to basal medium (3/4 strength MS salts and MS vitamins) improved the multiplication phase, reducing leaf chlorosis and raising the percentage of elongated shoots. The multiplication rate varied between 1.7 and 3 every month. Rooting percentage too was higher in the presence of charcoal, even when auxin was not added to the culture medium. The addition of 8.87 μM of benzyladenine and/or 1.44 or 2.88 μM of gibberellic acid did not influence significantly the multiplication, nor modifications in the FeSO4, MnSO4, CaCl2 content of the medium. The system described allows the multiplication, elongation and rooting of black wattle in one step.