Molecular chaperones, including the heat-shock proteins (Hsps), are a ubiquitous feature of cells in which these proteins cope with stress-induced denaturation of other proteins. Hsps have received the most attention in model organisms undergoing experimental stress in the laboratory, and the function of Hsps at the molecular and cellular level is becoming well understood in this context. A complementary focus is now emerging on the Hsps of both model and nonmodel organisms undergoing stress in nature, on the roles of Hsps in the stress physiology of whole multicellular eukaryotes and the tissues and organs they comprise, and on the ecological and evolutionary correlates of variation in Hsps and the genes that encode them. This focus discloses that (a) expression of Hsps can occur in nature, (b) all species have hsp genes but they vary in the patterns of their expression, (c) Hsp expression can be correlated with resistance to stress, and (d) species' thresholds for Hsp expression are correlated with levels of stress that they naturally undergo. These conclusions are now well established and may require little additional confirmation; many significant questions remain unanswered concerning both the mechanisms of Hsp-mediated stress tolerance at the organismal level and the evolutionary mechanisms that have diversified the hsp genes.

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Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology

Gel electrophoresis of proteins has become a standard and powerful research tool for application in a multitude of biological disciplines One form of protein electrophoresis, isozyme analysis, has become particularly prominent in systematic and evolutionary biology as well as agronomy (Tanksley and Orton, 1983) Isozymes, or multiple molecular forms of enzymes, are enzymes that share a common substrate but differ in electrophoretic mobility (Markert and Moller, 1959) They are revealed when tissue extracts are subjected to electrophoresis in various types of gels and subsequently submersed in solutions containing enzyme-specific stains Genetic analysis may indicate that some of the variant electromorphs are encoded by alternate alleles at a single locus, in which case the allelic products are termed allozymes (Prakash et al, 1969) Data retrieved from electrophoretic gels consist of the number and relative mobilities of various enzyme products, which with appropriate genetic analyses become transformed into single or multilocus genotypes for each individual analyzed Reasons are many for the popularity of electrophoretic data (Avise, 1975; Gottlieb, 1977; Crawford, 1983), but foremost among these is that isozymes provide a series of readily scored, single-gene markers

Visualization and Interpretation of Plant Isozymes

The ability to observe allelic variation at isozyme loci has revolutionized research in the fields of biochemical genetics, population genetics, and evolution. This variation, called allozymic polymorphism, has been used in plants to examine genetic processes at every stage of the life cycle and to ascertain genetic diversity in all major crops as well as many other species. Yet the potential for using allozymes as genetic markers was not immediately predicted when isozyme variability was initially described (Hunter and Marker, 1957; Markert and Moller, 1959). In fact, the extent and prevalence of allozyme polymorphism was a rather disconcerting surprise to evolutionary biologists. Classical evolutionary theory had predicted that the most “efficient” form of an enzyme should, over time, become predominant in isolated populations, with an occasional rare allele produced through mutation. The discovery in many populations of relatively high levels of polymorphism at isozyme loci has forced a major reconsideration of evolutionary theory (Kimura and Crow, 1964; Koehn et al., 1983; Kimura, 1983).

Genetics of Plant Isozymes

We present a genome-wide view of the male gametophytic transcriptome in Arabidopsis based on microarray analysis. In comparison with the transcriptome of the sporophyte throughout development, the pollen transcriptome showed reduced complexity and a unique composition. We identified 992 pollen-expressed mRNAs, nearly 40% of which were detected specifically in pollen. Analysis of the functional composition of the pollen transcriptome revealed the over-representation of mRNAs encoding proteins involved in cell wall metabolism, cytoskeleton, and signaling and under-representation of mRNAs involved in transcription and protein synthesis. For several gene families, we observed a common pattern of mutually exclusive gene expression between pollen and sporophytic tissues for different gene family members. Our results provide a 50-fold increase in the knowledge of genes expressed in Arabidopsis pollen. Moreover, we also detail the extensive overlap (61%) of the pollen transcriptome with that of the sporophyte, which provides ample potential to influence sporophytic fitness through gametophytic selection.

Comparative Analysis of the Arabidopsis Pollen Transcriptome

In the primitive angiosperms, closed carpels are believed to have evolved as protection for ovules, which would otherwise be injured by animal pollinators. The hypothesis is presented that, whatever the origin and other functions of angiosperms, insect pollination and closed carpels may, in combination, reduce the influence of random variation on pollen tube competition, thus enhancing the ability of natural selection to act on the gametophytic phase of the life cycle. The microgametophytic phase represented by vast numbers of haploid individuals can then serve, by insect pollination and closed carpels, as a screen against any genome not functioning with a high degree of metabolic vigor. Poorly balanced genomes could thus be eliminated at relatively little cost. Insect-pollinated angiosperms would therefore benefit from positive aspects of sexual recombination. Such a system may have allowed the angiosperms to undergo their rise to dominance.

http://science.sciencemag.org/content/sci/206/4414/20.full.pdf

The rise of the angiosperms: a genecological factor.

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.

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

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.

The extent of gametophytic-sporophytic gene expression 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.

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

There is evidence that male gametophyte selection is a widespread phenomenon in higher plants. The pollen tube growth rate is one of the main components of gametophyte selective value; genetic variability for this trait, due to the effect of single genes or to quantitative variation, has been described in maize. However, indication of gametophytic selection has been indirectly obtained; its effect was revealed by the positive relation observed between gametophyte competitive ability and sporophyte metrical traits. This paper considers the results of selection applied to gametophyte populations produced from single plants. The competitive ability of the lines was evaluated in comparison with that of a standard line by means of the pollen mixture technique. Sporophytic traits were measured in the hybrid progeny obtained by crossing selected S3 and S4 families with an unrelated single cross and an inbred line. Gametophyte selection produced inbred lines with high gametophyte competitive ability. In view of the selection procedure adopted, this result was interpreted as an indication of haploid expression of genes involved in the control of pollen tube growth. Moreover, this gametophytic trait was positively correlated with sporophytic traits (seedling weight, kernel weight and root tip growth in vitro), indicating that both groups of characters have a common genetic basis.

Male gametophytic selection in maize.

In order to measure differences in the pollen growth rate of numerous lines of maize and to investigate the main features of their genetic control, gametophyte growth was studied in vitro and in vivo. 
In vitro pollen tube growth of twenty inbred lines and seven hybrids was measured; a remarkable variability was observed in the growth rate of the inbred lines examined: most lines were distinct, showing different levels of growth. Analysis of frequency distribution of pollen tube lengths for pairs of inbred lines and their F1′ s revealed greater variance among lengths of F1 pollen tubes, presumably indicating the segregation of genetic factors expressed in the gametophyte. Similar frequency distributions of tube lengths in pollen produced by two pairs of reciprocal hybrids virtually excluded the presence of a cytoplasmic component. 
In vivo competitive ability of pollen tubes was measured as the increase in relative fertilization frequency from apex to base of the ear. Mixtures were made using two types of genetically distinguishable pollen, and were applied to a female common parent. Nine pairs of inbred lines furnished the pollen for the mixtures. In all cases where the B14 line was involved, this pollen type fertilized nearly all the ovules, perhaps indicating the presence of a gametophytic factor. When other lines were compared, the ears contained mixtures of the two possible seed types, the relative proportions of which indicated the differential competitive abilities of the two pollen tube types. A comparison between in vitro and in vivo behavior was made for some genotypes. In vivo results generally agreed with in vitro results. The degree of the differences between lines however was changed, presumably because pollen-style or pollen-pollen interactions are absent in vitro. Differing growth patterns between lines were also revealed in vivo by direct observation of fluorescent pollen tubes within the silks, a finding which may be useful in further studies.

M. Sari Gorla

Papers

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

The extent of gametophytic-sporophytic gene expression in maize.

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

Male gametophytic selection in maize.

Genetic variability of gametophyte growth rate in maize.