Organisation and structural evolution of the rice glutathione S-transferase gene family.

Glutathione S-transferases (GSTs) comprise a large family of key defence enzymes against xenobiotic toxicity. Here we describe the comprehensive characterisation of this important multigene family in the model monocot species rice [ Oryza sativa(L.)]. Furthermore, we investigate the molecular evolution of the family based on the analysis of (1) the patterns of within-genome duplication, and (2) the phylogenetic relationships and evolutionary divergence among rice, Arabidopsis, maize and soybean GSTs. By in-silico screening of the EST and genome divisions of the Genbank/EMBL/DDBJ database we have isolated 59 putative genes and two pseudogenes, making this the largest plant GST family characterised to date. Of these, 38 (62%) are represented by genomic and EST sequences and 23 (38%) are known only from their genomic sequences. A preliminary survey of EST collections shows a large degree of variability in gene expression between different tissues and environmental conditions, with a small number of genes (13) accounting for 80% of all ESTs. Rice GSTs are organised in four main phylogenetic classes, with 91% of all rice genes belonging to the two plant-specific classes Tau (40 genes) and Phi (16 genes). Pairwise identity scores range between 17 and 98% for proteins of the same class, and 7 and 21% for interclass comparisons. Rapid evolution by gene duplication is suggested by the discovery of two large clusters of 7 and 23 closely related genes on chromosomes 1 and 10, respectively. A comparison of the complete GST families in two monocot and two dicot species suggests a monophyletic origin for all Theta and Zeta GSTs, and no more than three common ancestors for all Phi and Tau genes.

A multivariate approach to the problem of QTL localization.

QTL mapping with statistical likelihood-based procedures or asymptotically equivalent regression methods is usually carried out in a univariate way, even if many traits were observed in the experiment. Some proposals for multivariate QTL mapping by an extension of the maximum likelihood method for mixture models or by an application of the canonical transformation have been given in the literature. This paper describes a method of analysis of multitrait data sets, aimed at localization of QTLs contributing to many traits simultaneously, which is based on the linear model of multivariate multiple regression. A special form of the canonical analysis is employed to decompose the test statistic for the general no-QTL hypothesis into components pertaining to individual traits and individual, putative QTLs. Extended linear hypotheses are used to formulate conjectures concerning pleiotropy. A practical mapping algorithm is described. The theory is illustrated with the analysis of data from a study of maize drought resistance.

Identification of genetic factors for Alachlor tolerance in maize by molecular markers analysis.

Genetic factors controlling tolerance to the herbicide Alachlor in maize were localised by means of two different strategies. In the first approach, backcross (BC) plants, derived from pollen which had been subjected to selective pressure for resistance to the herbicide, were analysed for segregation distortion at 47 RFLP loci and compared to BC plants obtained from non-selected pollen. Preferential transmission of five chromosomal regions where putative QTLs (Quantitative Trait Loci) are localised was revealed in the BC plants from selected pollen. A second approach was based on a classical linkage analysis for segregation of the same set of RFLPs and factors controlling the trait, in a BC population of 210 individuals, by means of regression analysis. This study detected seven significant loci in four genomic regions. Overall, two loci revealed both segregation distortion and association with the expression of the trait, indicating linkage to genes expressed in both gametophytic and sporophytic phase. Three chromosomal regions appeared to carry factors involved in plant tolerance to Alachlor which are not expressed in pollen. Conversely, three loci were linked to factors selectable in pollen, but did not reveal significant association with tolerance in the plant in the segregating populations.

Molecular analysis and mapping of two genes encoding maize glutathione S-transferases (GST I and GST II).

Maize glutathione S-transferase (GST) isozymes are encoded by a gene family comprising at least five genes, three of which (Gst I, II and III) have recently been isolated and sequenced. The enzymes are active as homo or heterodimers and exhibit intraspecific polymorphism including a "null" variant for the two major isoforms expressed in roots. Northern blot analyses performed on total root RNA from "null" and "plus" genotypes, using Gst I- and Gst II-specific probes, indicated that the Gst I gene controls the expression of the two major GST isoforms expressed in roots. Gst I and Gst II were mapped by RFLP analysis using an F2 population of 149 individuals previously characterized. Gst I was localized on the long arm of chromosome 8, while two putative Gst II loci were mapped to chromosome 8 (70 cM from Gst I) and 10, respectively.

Quantitative trait loci (QTLs) for pollen thermotolerance detected in maize.

Pollen thermotolerance is an important component of the adaptability of crops to high temperature stress. The tolerance level of the different genotypes in a population of 45 maize recombinant inbred lines was determined as the degree of injury caused by high temperature to pollen germinability (IPGG) and pollen tube growth (IPTG) in an in vitro assay. Both traits revealed quantitative variability and high heritability. The traits were genetically dissected by the analysis of molecular markers using 184 mapped restriction fragment length polymorphisms (RFLPs). Significant genetic correlation between the markers and the trait allowed us to identify a minimum number of five qualitative trait loci (QTLs) for IPGG and six QTLs for IPTG. Their chromosomal localization indicated that the two characters are controlled by different sets of genes. In addition, IPGG and IPTG were shown to be basically independent of the pollen germination ability and pollen tube growth rate under non-stress conditions. These results are discussed in relation to their possible utilization in a breeding strategy for the improvement of thermotolerance in maize.

Sporophytic response to pollen selection for Alachlor tolerance in maize.

In order to assess the efficiency of male gametophytic selection (MGS) for crop improvement, pollen selection for tolerance to herbicide was applied in maize. The experiment was designed to test the parallel reactivity to Alachlor of pollen and plants grown in controlled conditions or in the field, the response to pollen selection in the sporophytic progeny, the response to a second cycle of MGS, and the transmission of the selected trait to the following generations. The results demonstrated that pollen assay can be used to predict Alachlor tolerance under field conditions and to monitor the response to selection. A positive response to selection applied to pollen in the sporophytic progeny was obtained in diverse genetic backgrounds, indicating that the technique can be generally included in standard breeding programs; the analysis of the data produced in a second selection cycle indicated that the selected trait is maintained in the next generation.

Quantitative expression of maize HSPs: genetic dissection and association with thermotolerance.

In higher plants, within-species qualitative polymorphism for heat shock proteins (HSPs) is extremely rare, even between genotypes showing different heritable levels of thermotolerance. Here we have explored the amount of quantitative variability in HSP synthesis in maize. We have analyzed the quantitative expression of the typical HSPs in a set of recombinant inbreds (RIs) derived from the f1 hybrid between a thermotolerant (T232)- and a thermosensitive (CM37)-genotype, characterized for about 200 mapped RFLP loci. Significant differences were detected in the level of expression of five HSPs, and their frequency distribution in the RI population is that of a quantitative trait. Subsequent mapping of loci controlling the characters, based on RFLP analysis, confirmed the multigenic control of HSP expression: the regression analysis of the band intensities of each variant HSP on RFLPs revealed, for the different HSPs, a minimum number of three to eight quantitative trait loci (QTLs) accounting for a high proportion (0.35-0.60) of the genetic variability of these bands. An analysis of the correlation between the variability of HSPs and that of cellular membrane stability, a cellular component of thermotolerance, did not reveal any significant association of the two parameters.

Molecular markers (RFLPs and HSPs) for the genetic dissection of thermotolerance in maize.

Cellular membrane stability (CMS) is a physiological index widely used to evaluate thermostability in plants. The genetic basis of the character has been studied following two different approaches: restriction fragment length polymorphism (RFLP) analysis, and the effects of segregating heat shock protein (HSP) loci. RFLP analysis was based on a set of recombinant inbreds derived from the T32 × CM37 F1 hybrid and characterized for about 200 RFLP loci. Heritability of CMS estimated by standard quantitative analysis was 0.73. Regression analysis of CMS on RFLPs detected a minimum number of six quantitative trait loci (QTL) accounting for 53% of the genetic variability. The analysis of the matrices of correlation between RFLP loci, either within or between chromosomes, indicates that no false assignment was produced by this analysis. The effect of HSPs on the variability of the CMS was tested for a low-molecular-weight peptide (HSP-17) showing presence-absence of segregation in the B73 × Pa33 F2 population. Although the genetic variability of the character was very high (h (2)=0.58) the effect of HSP-17 was not significant, indicating either that the polypeptide is not involved in the determination of the character or that its effect is not statistically detectable.

Androstane derivative devoid of anabolic-virilizing effects and endowed with an antiglucocorticoid activity.

2-Carbodecyloxy-17 alpha-methylandrosta-1,4-dien-11 beta,17 beta-dihydroxy-3-one (decylroxibolone, BR 917) is a new androstane derivative, esterified with decyl alcohol, carrying a methyl group in the 17 position, a hydroxyl group in the 11 beta position and a carboxyl group in position 2. Unlike norandrostenolone decanoate, decylroxibolone did not cause any weight increase of the levator ani muscle and of the seminal vesicles in castrated rats, nevertheless exerting a marked antiglucocorticoid activity. This new steroid agent can consequently act positively on the nitrogen metabolism, being concurrently devoid of undesired virilizing anabolic effect.

Androstane derivatives--roxibolone and decylroxibolone--devoid of any affinity for the androgenic prostate and muscle receptors.

Unlike testosterone propionate and norandrostenolone decanoate, some new androstane derivatives did not cause any weight increase of the levator ani muscle and of the seminal vesicles in castrated rats. Actually, these new androsterone derivatives, characterized by the presence of a carboxyl group in the position 2 and a hydroxyl group in the 11 alpha or beta position, have entirely lost their affinity for the androgenic receptors of the prostate and of the skeletal muscles.

Nesosteine: a new mucoregulatory agent.

A new, original molecule, nesosteine, modified both the rheology and the production of tracheobronchial mucus in rabbits. The drug highly significantly reduced the viscosity of tracheobronchial mucus in animals made bronchitic by H2SO4 aerosol and markedly increased mucoproduction in healthy animals. Nesosteine was more active than the best known mucolytic/mucoregulatory drugs, such as sobrerol, N-acetylcysteine, bromexine, ambroxol, S-carboxymethylcysteine and mercaptopropionylglycine. The fluidifying activity of the drug was also demonstrated in vitro (pig's gastric mucin), although this proved to be less marked than in vivo. Nesosteine reduced the amount of total proteins of the tracheobronchial mucus, acting on albumin, alpha 1, alpha 2, beta and gamma mucoproteins.

The extent of gametophytic-sporophytic gene expression in maize.

To determine the extent of gametophytic gene expression and the type of transcription, haploid or haplo-diploid, of the genes, isozymes were used as genetic markers. Fifteen enzymatic systems, including thirty-four isozymes, were studied. The determination of the type of expression of genes coding for multimeric enzymes was based on the comparison of electrophoretic patterns of pollen and of sporophytic tissues from plants heterozygous for electrophoretic mobility: if gene expression in pollen is of a gametophytic (haploid) origin, pollen, unlike the sporophyte, would reveal only the parental homomultimeric bands. The enzymes analyzed can be grouped in three categories according to type of gene expression: i) enzymes present in both pollen and sporophyte, coded by the same gene with haplo-diploid expression; ii) enzymes controlling analogous functions in pollen and sporophyte, coded by different genes, expressed in only one of the two phases; iii) enzymes present in two or more forms in the sporophyte and only in one form in the gametophyte. The data allow the proportion of haplo-diploid gene expression in the loci examined to be estimated at 0.72; 0.22 and 0.06 being the proportions attributable to the sporophytic and gametophytic domains, respectively.

Haplodiploid gene expression in maize and its detection.

A method for the demonstration of the gametophytic origin of genetic variability in maize is described. For genes coding dimeric or multimeric enzymes, haploid expression can be demonstrated by means of translocations between A and B chromosomes (TB-A), which make it possible to obtain hyperploid pollen grains, partially diploid and heterozygous for electrophoretic mobility. Comparison of the electrophoretic pattern of this pollen type (three bands) and that of normal grains produced by a heterozygous F/S plant (two bands only) reveals haploid transcription of the monomeric forms. The procedure was tested on ADH-1 and used to demonstrate haploid expression for GOT-1. The data obtained suggest, moreover, that the reduction in male gamete transmission of duplications may be due to differences in pollen competitive ability rather than to processes affecting microspore maturation.