Intron retention regulates the expression of pectin methyl esterase inhibitor (Pmei) genes during wheat growth and development.

Pectin is an important component of the plant cell wall and its remodelling occurs during normal plant growth or following stress responses. Pectin is secreted into the cell wall in a highly methyl-esterified form and subsequently de-methyl-esterified by pectin methyl esterase (PME), whose activity is controlled by the pectin methyl esterase inhibitor protein (PMEI). Cereal cell wall contains a low amount of pectin; nonetheless the level and pattern of pectin methyl esterification play a primary role during development or pathogen infection. Since few data are available on the role of PMEI in plant development and defence of cereal species, we isolated and characterised three Pmei genes (Tdpmei2.1, Tdpmei2.2 and Tdpmei3) and their encoded products in wheat. Sequence comparisons showed a low level of intra- and inter-specific sequence conservation of PMEIs. Tdpmei2.1 and Tdpmei2.2 share 94% identity at protein level, but only 20% identity with the product of Tdpmei3. All three Tdpmei genes code for functional inhibitors of plant PMEs and do not inhibit microbial PMEs or a plant invertase. RT-PCR analyses demonstrated, for the first time to our knowledge, that Pmei genes are regulated by intron retention. Processed and unprocessed transcripts of Tdpmei2.1 and Tdpmei2.2 accumulated in several organs, but anthers contained only mature transcripts. Tdpmei3 lacks introns and its transcript accumulated mainly in stem internodes. These findings suggest that products encoded by these Tdpmei genes control organ- or tissue-specific activity of specific PME isoforms in wheat.

Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors.

We have identified, expressed and characterized two genes from Arabidopsis thaliana (AtPMEI-1 and AtPMEI-2) encoding functional inhibitors of pectin methylesterases. AtPMEI-1 and AtPMEI-2 are cell wall proteins sharing many features with the only pectin methylesterase inhibitor (PMEI) characterized so far from kiwi fruit. Both Arabidopsis proteins interact with and inhibit plant-derived pectin methylesterases (PMEs) but not microbial enzymes. The occurrence of functional PMEIs in Arabidopsis indicates that a mechanism of controlling pectin esterification by inhibition of endogenous PMEs is present in different plant species.

Extracellular H(2)O(2) induced by oligogalacturonides is not involved in the inhibition of the auxin-regulated rolB gene expression in tobacco leaf explants.

alpha-1,4-Linked oligogalacturonides (OGs) inhibit auxin-regulated transcriptional activation of a rolB-beta-glucuronidase (GUS) gene fusion in tobacco (Nicotiana tabacum) leaf explants (D. Bellincampi, M. Cardarelli, D. Zaghi, G. Serino, G. Salvi, C. Gatz, F. Cervone, M. M. Altamura, P. Costantino, G. De Lorenzo [1996] Plant Cell 8: 477-487). In this paper we show that inhibition by OGs is very rapid, with a short lag time, and takes place even after rolB promoter activation has initiated. OGs also induce a transient and catalase-sensitive accumulation of H(2)O(2) in the leaf explant culture medium. OGs with a degree of polymerization from 12 to 15 are required for both the inhibition of the auxin-induced rolB-driven accumulation of GUS and the induction of H(2)O(2) accumulation(.) However, OG concentration for half-maximal induction of H(2)O(2) accumulation is approximately 3-fold higher than that for half-maximal inhibition of rolB promoter activity. The inhibition of rolB promoter activity is not influenced by the addition of catalase or superoxide dismutase, suggesting that H(2)O(2) and superoxide are not involved in this effect. A fungal oligo-beta-glucan elicitor induces extracellular H(2)O(2) accumulation at comparable or higher levels than those observed with OGs, but does not prevent the auxin-induced accumulation of GUS. We conclude that H(2)O(2) produced upon treatment with OGs is not involved in the inhibition of the auxin-induced expression of the rolB gene.

Oligogalacturonides Prevent Rhizogenesis in rolB-Transformed Tobacco Explants by Inhibiting Auxin-Induced Expression of the rolB Gene.

Oligogalacturonides elicit several defense responses and regulate different aspects of growth and development in plants. Many of the development-related effects of oligogalacturonides appear to be amenable to an auxin antagonist activity of these oligosaccharins. To clarify the role of oligogalacturonides in antagonizing auxin, we analyzed their effect on root formation in leaf explants of tobacco harboring the plant oncogene rolB. We show here that oligogalacturonides are capable of inhibiting root morphogenesis driven by rolB in transgenic leaf explants when this process requires exogenous auxin. Because rolB expression is induced by auxin and dramatically alters the response to this hormone in transformed plant cells, the inhibiting effect of oligogalacturonides could be exerted on the induction of rolB and/or at some other auxin-requiring step(s) in rhizogenesis. We show that oligogalacturonides antagonize auxin primarily because they strongly inhibit auxin-regulated transcriptional activation of a rolB-[beta]-glucuronidase gene fusion in both leaf explants and cultured leaf protoplasts. In contrast, oligogalacturonides do not inhibit rhizogenesis when rolB transcriptional activation is made independent of auxin, as shown by the lack of inhibition of root formation in leaf explants containing rolB driven by a tetracycline-inducible promoter.

Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA.

Single and multiple infections of carrot discs were carried out with Agrobacterium strains harbouring different segments of pRi1855 TL-DNA cloned in the binary vector Bin 19 and with a strain carrying the TR-DNA from the same Ri plasmid. Roots induced by the various co-inoculations were cultured and their growth patterns were followed. Abundant roots could be induced by TL-DNA rol genes A, B and C as a single insert (rolA + B + C) and by rolB alone provided an extended segment beyond its 5' non-coding region was included in the construction. A depression of rooting capability was caused by the inclusion of rolC together with rolB (rolB + C). In all cases co-inoculation with the Agrobacterium carrying TR-DNA-borne auxin genes was necessary for root induction since none of the rol constructions was in itself capable of eliciting any response; an exceeding majority of these roots were however shown to contain rol genes but no TR-DNA. Rooting was also elicited if rol constructions were co-inoculated with a strain carrying TL-DNA genes 13 and 14 (ORF13 + 14) instead of the TR-DNA strain. These roots were shown to contain both rol genes and ORF13 + 14. Striking differences in growth properties were shown by roots containing different complements of TL-DNA genes. Typical hairy root traits, high growth rate, branching and, most noticeably, absence of geotropism, were shown by roots containing rolB alone, while roots with rolA + B + C were geotropic as normal carrot roots. Hairy root traits were conferred to rolA + B + C roots by the concomitant presence of ORF13 + 14 and by the addition of auxin to the culture medium. A model is presented which attempts to rationalize the growth patterns by assigning interplaying roles to the various TL-DNA genes involved.

A comparative study on selected chemical carcinogens for chromosome malsegregation, mitotic crossing-over and forward mutation induction in Aspergillus nidulans.

10 "false negative" chemical carcinogens, i.e. ineffective in bacterial mutagenicity assays, were thoroughly investigated for their genotoxic activity in the mould Aspergillus nidulans. Forward mutations (methionine suppressors), mitotic crossing-over and chromosome malsegregation were the end-points scored. Positive results were obtained in tests for the induction of mitotic segregation with benzene, ethylenethiourea and urethane, which increased the frequency of abnormal presumptive aneuploid colonies with euploid sectors showing whole chromosome segregation (i.e. non-disjunctional diploids and haploids). The same compounds were ineffective in increasing the frequency of mitotic crossing-over or forward mutations. The other chemical carcinogens investigated, namely acetamide, amitrole, dieldrin, heptachlor epoxide, nitrilotriacetic acid, p,p'-DDT and thiourea were ineffective both as inducers of forward mutations and mitotic segregation.

High plating efficiency with plant cell cultures.

In this paper we describe a simple method to improve the plating efficiency in plant cell cultures.Two-stage plating is used; in the first stage the cells are inoculated at high density in 0.2% agarized culture medium for ten days to facilitate growth; under this condition, each cell produces a single micro-colony trapped in the agar network. In the second stage the colonies are plated at different densities in 1% agarized medium.These colonies are self-sufficient and able to improve the cell growth by conditioning the medium.

Induced gene mutation and mitotic non-disjunction in A. nidulans.

Toxicants of different classes were analysed for capacity to induce gene mutation and mitotic non-disjunction in Aspergillus nidulans, using selective and permissive tests, respectively. Ethanol, Amphotericin B and MIcanozole, all affecting membrane integrity, induced only non-disjunction, emphasizing the röle of the membrane in mitosis. Benomyl and isopropyl-3-chlorophenyl carbamate (CIPC), two pesticides which interfere with spindle system, induced only non-disjunction. Conversely, mitomycin C markedly increased mutation rate but not ono-disjunction and scarcely affected the viability. The comparative analysis of these two different genetic damages should prove useful in evaluating hazards of drugs.

MMS induction of different types of genetic damage in Aspergillus nidulans: a comparative analysis in mutagenesis.

Methyl methanesulphonate (MMS) was used to test the induction of gene mutation, somatic crossing-over and mitotic non-disjunction in A. nidulans. Gene mutation was tested by inducing mutants resistant to 8-azaguanine and revertants of methG1 in a haploid strain. Somatic crossing-over was tested in heterozygous diploids, both with a selective method, i.e. inducing homozygosis to FPA resistance in a heterozygous fpa A1/+ strain, and with a non-selective method, i.e. identifying the frequencies of colour sectors. This latter method was also used to estimate the induction of non-disjunction because additional markers were present which permitted us to distinguish the two types of colour segregant. Generally, 3 different experimental procedures were used, namely the "plate test", i.e. plating of conidia in agar media containing MMS, and two types of "liquid test", i.e. brief treatment of quiescent or pre-germinated conidia in MMS solution before they were plated on agar media. Point mutations were induced with about equal efficiency with each method, whereas crossing-over was induced preferentially when germinating conidia were exposed to MMS. On the other hand, non-disjunction was induced in germinating and quiescent spores with equal efficiency, but such segregants were not recovered with the selective (fpa) method. The results are discussed for both their practical use in the mutagenic testing procedure and their theoretical implication.

Analysis of mitotic nondisjunction with Aspergillus nidulans.

Two methods to detect the induction of nondisjunction with a diploid stable strain of A. nidulans are described. The first method gives only qualitative results, while the second method is quantitative and dose-effect curves can be done. Some physiological parameters affecting the induction of nondisjunction can also be studied, because either quiescent or germinating conidia can be treated with the drug under test. Some agents inducing nondisjunction were also tested for the induction of point mutation and somatic crossing-over with these comparative analysis. Two classes of agents inducing nondisjunction may be detected: the first causes all possible types of genetic damage either on quiescent or germinating conidia (a representative of this class is MMS) and acts presumably on the DNA level; the second acts only on germinating conidia and does not produce point mutation or crossing over. A representative of this class is Benomyl which interferes with spindle microtubules. A list of compounds tests is included.