Characterization of a ubiquitous expressed gene family encoding polygalacturonase in Arabidopsis thaliana.

Pectin, as one of the major components of plant cell wall, has been implicated in many developmental processes occurring during plant growth. Among the different enzymes known to participate in the pectin structure modifications, polygalacturonase (PG) activity has been shown to be associated with fruit ripening, organ abscission and pollen grain development. Until now, sequence analyses of the deduced polypeptides of the plant PG genes allowed their grouping into three clades corresponding to genes involved in one of these three activities. In this study, we report the sequence of three genomic clones encoding PG in Arabidopsis thaliana. These genes, together with 16 other genes present in the databases form a large gene family, ubiquitously expressed, present on the five chromosomes with at least two gene clusters on chromosomes II and V, respectively. Phylogenetic analyses suggest that the A. thaliana gene family contains five classes of genes, with three of them corresponding to the previously defined clades. Comparison of positions and numbers of introns among the A. thaliana genes reveals structural conservation between genes belonging to the same class. The pattern of intron losses that could have given rise to the PG gene family is consistent with a mechanism of intron loss by replacement of an ancestral intron-containing gene with a reverse-transcribed DNA copy of a spliced mRNA. Following this event of intron loss, the acquisition of introns in novel positions is consistent with a mechanism of intron gain at proto-splice sites.

Differential expression of a polygalacturonase gene family in Arabidopsis thaliana.

By systematic sequencing of a flower bud cDNA library from Arabidopsis thaliana, we have identified four cDNAs encoding polygalacturonase. The corresponding genes, together with seven other A. thaliana genes present in the databases, form a small gene family. Sequence comparisons of the deduced polypeptides within the gene family or with other plant polygalacturonases allow classification of the genes into different clades. Five polygalacturonases, including all those isolated from the flower buds, are closely related to the enzyme in pollen. Of the six remaining polygalacturonases, three are more closely related to the abscission-specific type of enzyme and two others to the fruit polygalacturonase. The last one is more distantly related to the others and might correspond to a new type of polygalacturonase. Expression of the different genes was analysed on Northern blots and by a PCR-based strategy. Results indicate that if, as expected, the cDNAs isolated from the flower bud library are strongly expressed in pollen, other genes are expressed at a low level in young developing tissues, such as in seedlings and roots, suggesting that they could be implicated in the cell wall modifications observed during cell elongation and/or expansion which occur in these tissues.

RGD-dependent growth of maize calluses and immunodetection of an integrin-like protein.

When maize calluses are grown in the presence of the RGD peptide, important morphological changes are observed indicating the presence of a likely RGD-binding receptor. Polyclonal antibodies generated against the human beta1 integrin subunit, the platelet integrin alphaIIbeta3 (P23) and antibodies specific for either the beta3 platelet chain or the alphaIIb polypeptide cross-react with glycoproteins in Western blot analyses. Immunoprecipitation assays indicate that this maize integrin-like protein shares structural similarities with the animal alphaIIbeta3 complex. We also show that AcAt2, a polyclonal antibody raised against Arabidopsis proteins purified on an RGD column, interacts with a maize protein.

A plant surface protein sharing structural properties with animal integrins.

Using a polyclonal antibody (P23) generated against the human platelet integrin aIIb beta3 and a FITC-conjugate secondary antibody, fluorescence is observed at the surface of protoplasts isolated from Arabidopsis thaliana and Rubus fruticosus. Arabidopsis thaliana cells grown in suspension culture containing P23 and glycylarginylglycylaspartylserine (GRGDS), a synthetic peptide containing the RGD sequence found in many extracellular matrix adhesive proteins demonstrated aberrant cell wall/plasma membrane interactions and organization. When glycoproteins from these plants, purified on a concanavalin A Sepharose 4B, were subjected to SDS/PAGE and Western blotting, under reduced and non-reduced conditions, immunoblots probed with P23 revealed bands in both species. A shift in electrophoretic mobility is observed to different apparent molecular mass when no reducing agent is present. When purified by immunoaffinity chromatography on anti-aIIb beta3 Sepharose or Sepharose linked to the synthetic peptide D-Arg-Gly-Asp-Trp, the major antigenic components detected migrate at 30 kDa and 60 kDa in the first experiment and 60 kDa in the second one. Only the 60-kDa component is immunodetected with antibodies specific for either the beta3 platelet chain or the aIIb polypeptide, suggesting the presence of two polypeptides co-migrating. To address more precisely the structure of this complex in plants, competition assays were performed. A significant inhibition is observed with CS3 a monoclonal antibody that interacts with the complexed form aIIb beta3 but not the dissociated subunits. Further structural similarities with the animal aIIb beta3 complex is demonstrated with Western blotting detection after plant glycoproteins immunoprecipitation with CS3 in absence or presence of 5 mM EDTA to dissociate the complex. We also present data on the characterization of a polyclonal antibody, named AcAt2, raised against Arabidopsis glycocoproteins purified by affinity chromatography on a D-RGDW column and eluted with the same peptide, that specifically interacts with the animal aIIb beta3 receptor.

The ubiquitous presence of exopolygalacturonase in maize suggests a fundamental cellular function for this enzyme.

Exopolygalacturonase (exoPG) is a pectin-degrading enzyme abundant in maize pollen. Using immunochemistry and in situ hybridization it is shown that in addition to its presence in pollen, exoPG is also present in sporophytic tissues, such as the tapetum and mesophyll cells. The enzyme is located in the cytoplasm of pollen and of some mesophyll cells. In other mesophyll cells, the tapetum and the pollen tube, exoPG is located in the cell wall. The measurement of enzyme activity shows that exoPG is ubiquitous in the vegetative organs. These results suggest a general function for exoPG in cell wall edification or degradation. ExoPG is encoded by a closely related multigene family. The regulation of the expression of one of the exoPG genes was analyzed in transgenic tobacco. Reporter GUS activity was detected in anthers, seeds and stems but not in leaves or roots of transgenic plants. This strongly suggests that the ubiquitous presence of exoPG in maize is the result of the expression of different exoPG genes.

Expression of Chloroplast and Mitochondrial Genes during Microsporogenesis in Maize.

Mitochondrial and plastid gene expression has been examined during maize (Zea mays) microsporogenesis. Accumulation of transcripts was found for three mitochondrial genes studied (cob, atp6, and atp9) at the mid-term of pollen development. In contrast, these mitochondrial transcripts were undetectable in mature pollen. Southern and DNA gel blot experiments showed that the copy number of mitochondrial genes was amplified in microspores at stages preceding the accumulation of these transcripts. Plastid transcripts of the photosynthetic psbA and rbcL genes could not be detected after the two mitoses, whereas precursors of the 16S rRNA are detected at low levels.

Characterization of pollen polygalacturonase encoded by several cDNA clones in maize.

A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, has been isolated from a cDNA library using a differential screening method with cDNA probes representative of microspores at early or late developmental stages. The encoded 410 amino acid polypeptide has significant homology with various polygalacturonases (PG) described elsewhere. Two polypeptides, of 49 and 53 kDa respectively, have been identified in the active PG fraction, isolated from mature pollen by immuno-cross-reaction with tomato PG antibodies. According to their N-terminal sequence, they can be identified as being mature peptides encoded by the PG1 cDNA clone. We propose that these two proteins derive from a unique precursor through several post-translational events, including the excision of a 22 amino-terminal signal peptide and glycosylation. PG-encoding genes from a small genomic family. Sequence analysis of three PG cDNA clones shows that they are closely related. The divergence of nucleotides between these three cDNA clones is 1%. They encode the same product.

In vitro protein synthesis in isolated microspores of Zea mays at several stages of development.

A new procedure has been used providing large and homogenous populations of pollen from maize at different stages of their development. In order to label proteins synthesized during the course of microsporogenesis, a method has been developed that allows an efficient uptake of amino acids in the microspores. Results are presented showing that during pollen development three specific steps are involved: an early period active in protein synthesis, followed by a rest period when starch is accumulated, and a third period preceding the sorting out of mature pollen grains and during which protein synthesis starts again at a relatively low level. New polypeptides, some of which are very basic, appear at the time of starch deposition and accumulate up to the mature stage.

In vitro imaginal disc development and moulting hormone.

Imaginal leg and wing discs obtained from late-third-instar Drosophila larvae were cultured in vitro in various concentrations of ecdysterone ranging from 10(-10) to 10(-5) M in order to test the effect of hormone concentration on evagination and cell differentiation. At the optimal concentration of 8 x 10(-8) M discs evaginated normally, secreted the pupal cuticle, underwent apolysis, differentiated imaginal structures and secreted the imaginal cuticle. At suboptimal concentrations (10(-8) M and less), evagination was incomplete in a variable proportion of appendages. Morphogenetic movements were limited to the earlier ones; so that appendages did not emerge from the peripodial sac. Subsequent development, whenever it occurred, took place inside the peripodial sac. This particular type of 'endoevagination' was only obtained with sub-optimal hormone concentration. At supra-optimal concentrations (10(-6) M and more), evagination was always complete but further differentiation was inhibited. These results show that endoevagination is strictly related to insufficient supply of hormone and that morphogenesis and cell differentiation in imaginal discs are two independent phenomena, which respond to different levels of hormone stimulation.