Roman A. Volkov

Heat Stress- and Heat Shock Transcription Factor-Dependent Expression and Activity of Ascorbate Peroxidase in Arabidopsis

To find evidence for a connection between heat stress response, oxidative stress, and common stress tolerance, we studied the effects of elevated growth temperatures and heat stress on the activity and expression of ascorbate peroxidase (APX). We compared wild-type Arabidopsis with transgenic plants overexpressing heat shock transcription factor 3 (HSF3), which synthesize heat shock proteins and are improved in basal thermotolerance. Following heat stress, APX activity was positively affected in transgenic plants and correlated with a new thermostable isoform, APXS. This enzyme was present in addition to thermolabile cytosolic APX1, the prevalent isoform in unstressed cells. In HSF3-transgenic plants, APXS activity was detectable at normal temperature and persisted after severe heat stress at 44°C. In nontransgenic plants, APXS was undetectable at normal temperature, but could be induced by moderate heat stress. The mRNA expression profiles of known and three new Apx genes were determined using real-time PCR. Apx1 and Apx2 genes encoding cytosolic APX were heat stress and HSF dependently expressed, but only the representations of Apx2 mRNA met the criteria that suggest identity between APXS and APX2: not expressed at normal temperature in wild type, strong induction by heat stress, and HSF3-dependent expression in transgenic plants. Our data suggest thatApx2 is a novel heat shock gene and that the enzymatic activity of APX2/APXS is required to compensate heat stress-dependent decline of APX1 activity in the cytosol. The functional roles of modulations of APX expression and the interdependence of heat stress and oxidative stress response and signaling mechanisms are discussed.

Expression of the Apx gene family during leaf senescence of Arabidopsis thaliana

Oxygen-free radicals are thought to play an essential role in senescence. Therefore, the expression patterns of the small gene family encoding the H2O2 scavenging enzymes ascorbate peroxidase (APX; EC 1.11.1.11) were analyzed during senescence of Arabidopsis thaliana (L.) Heinh. Applying real-time RT-PCR, the mRNA levels were quantified for three cytosolic (APX1, APX2, APX6), two chloroplastic types (stromal sAPX, thylakoid tAPX), and three microsomal (APX3, APX4, APX5) isoforms identified in the genome of Arabidopsis. The genes of chloroplastic thylakoid-bound tAPX and the microsomal APX4 exhibit a strong age-related decrease of mRNA level in leaves derived from one rosette as well as in leaves derived from plants of different ages. In contrast to the tAPX, the mRNA of sAPX was only reduced in old leaves of old plants. The microsomal APX3 and APX5, and the cytosolic APX1, APX2, and APX6 did not show remarkable age-related changes in mRNA levels. The data show that expression of the individual APX genes is differentially regulated during senescence indicating possible functional specialization of respective isoenzymes. The hydrogen peroxide levels seem to be controlled very precisely in different cell compartments during plant development.

Small heat shock proteins are differentially regulated during pollen development and following heat stress in tobacco

In plants small heat shock proteins (sHsp) are abundantly expressed upon heat stress in vegetative tissue, however, sHsp expression is also developmentally induced in pollen. The developmental induction of sHsp has been related to the potential for stress-induced microspore embryogenesis. We investigated the polymorphism among sHsp and their expression during pollen development and after heat stress in tobacco. Real-time RT-PCR was used for quantification of mRNA of two known and nine newly isolated cDNAs representing cytosolic sHsp. At normal temperature most of these genes are not transcribed in vegetative tissues, however, all genes were expressed during pollen development. Low levels of mRNAs were found for sHsp-1A and -1B in early-unicellular stage, increasing four to sevenfold in mature pollen. Nine other genes are up-regulated in unicellular and down-regulated in bicellular pollen; three these genes show stage-specific expression. Western analysis revealed that cytosolic class I and II sHsp are developmentally expressed during all stages of pollen development. Different subsets of cytosolic sHsp genes are expressed in a stage-specific fashion suggesting that certain sHsp genes may play specific roles in early, others during later stages of pollen development. Heat stress results in a relatively weak and incomplete response in pollen: (i) the heat-induced levels of mRNA (excepting sHsp-2B, −3Cand -6) are much lower than in leaves, (ii) several sHsp are not detected after heat stress in pollen, although, they are heat-inducibly expressed in leaves. Application of heat stress, cold, and starvation, which induce microspore embryogenesis, modify mRNA levels and the patterns of 2-D-separated sHsp, but only heat stress enhances the expression of sHsp in microspores. There is no correlation of the expression of specific sHsp with the potential for microspore embryogenesis.

Ribosomal DNA in plant hybrids: Inheritance, rearrangement, expression

Abstract Ribosomal RNA genes (rDNA) represent a useful tool to study reticulate evolution. In allopolyploid plant species biparental or uniparental inheritance of 35S rDNA was demonstrated. Uniparental inheritance; as a result of differential elimination of one of the parental rDNA, can be accompanied by structural rearrangements of rDNA ofthe other parent and by formation of new rDNA variants. Particularly, homo‐genization of rDNA in allopolyploids appears to be an example of fast concerted evolution of repeated sequences. At the functional level, interaction between parental 35S rDNA loci in hybrids/allopolyploids leads to differential transcription/silencing, known as nucleolar dominance (ND). Depending on the combination of parental species, ND may be strong and stable, or weak, unstable and even reversible. Differential transcription of parental rDNA is regulated via cytosine methylation of DNA, histone modifications, and chromatin remodelling factors, but mechanisms providing discrimination of parental rDNA remain poorly understood. Probably, interplay between several factors such as local structural features of rDNA (especially subrepeated elements in the intergenic spacer region), unlinked loci, and the chro‐mosomal/genomic context determine ND. The ND and rDNA rearrangements reflect the dynamic nature and evolutionary plasticity of the genome in allopolyploids.

Plant highly repeated satellite DNA: Molecular evolution, distribution and use for identification of hybrids

Abstract Relationships among genomes are often revealed by the occurrence of common or related satellite DNA (satDNA) types. A typical satDNA characterized by specific sites for one (or more) restriction endonuclease(s) is called ‘restriction satellite DNA’. Restriction satDNA comprises ‐ in addition to transposons and retrotransposable elements ‐ often highly repeated genome components present in most higher plants. Large arrays of satDNA elements are concentrated at subtelo‐meric and/or centromeric regions (intermingled with other retrotransposon‐derived elements), however, they can be also located as large intercalating blocks along the chromosome. The head‐to‐tail tandemly arranged repeat units (monomers) of satDNA mostly exhibit lengths of 160 to 180 bp or 320 to 370 bp, but other lengths were also found in plants. In particular, in interspecific hybrids between more distantly related species, which often exist only after polyploidization, the individual repetitive DNA of the crossing partners contribute to recombination and rearrangement processes in the hybrids, thereby stimulating genome evolution. Here, we concentrate on the possible origin, molecular evolution, organization and distribution of highly repeated satDNA in various higher plants with emphasis on hybrids and allopolyploids.

Structural analysis of rDNA in the genus Nicotiana

The nucleotide sequence of the intergenic spacer (IGS) region between the 25S and the 18S rRNA coding regions has been determined for tobacco (Nicotiana tabacum). The IGS (5140 bp in length) can be subdivided into several regions (I–VII) two of which, upstream and downstream of the putative transcription initiation site (TIS), contain prominent subrepeats (A and C). The unique sequence in the central part of the IGS (region IV) preceding the TIS is extremely AT-rich. The distance from the putative TIS to the 5′ end of the 18S rRNA gene is 3005 bp. The IGS sequences are compared with potato (Solanum tuberosum) and tomato (Lycopersicon esculentum) IGS. Restriction mapping of 13 Nicotiana species shows that considerable rDNA repeat length heterogeneity in this genus is probably due to different numbers of A and C subrepeats.

Molecular Cell Biology: Organization and Molecular Evolution of rDNA, Nucleolar Dominance, and Nucleolus Structure

Recently, the ribosomes — molecularly well-characterized cell organelles where the process of translation of messenger RNA into polypeptides occurs — have attracted new interest since they can be considered as a huge ribozyme-like complex consisting of different proteins and RNA — the RNAs mainly fulfilling functional tasks in translation, the proteins serving more structural functions (Moore and Steitz 2002). Within the eukaryotic cell, two (animals and fungi) or three (plants) sites of a translational machinery are present, the cytoplasm, mitochondria and chloroplasts, and, respectively, the components are mostly encoded in the cell nucleus or the mitochondrial or chloroplast genome. Since new insights are gained into the production and the assembly of the ribosome components, ribosomal RNA (rRNA) and ribosomal proteins (r-proteins), in this chapter, we focus on the nucleolus, the site for cytoplasmic ribosome biogenesis and assembly occurring in the light-microscopically visible structure of the cell nucleus. Similarly as in prokaryotes, for all higher organisms two ribosome subunits are preformed in the nucleolus, which are evolutionary conserved: the small 40S subunit (SSU) containing 18S rRNA, and the large 60S subunit containing 25/28S plus 5.8S and 5S rRNA. After export of the ribosome subunits (LSU) into the cytoplasm, the functionally active ribosome is associated with translatable mRNA to an 80S complex corresponding to the 70S structure in chloroplasts and prokaryotic cells.

Molecular evolution of 5S rDNA of Solanum species (sect. Petota): application for molecular phylogeny and breeding

Abstract  Nucleotide sequences of 5S rRNA genes (5S rDNA) of 26 wild species of the genus Solanum (sect. Petota) originating from Middle or South America, four Solanum tuberosum breeding lines and one European species, Solanum dulcamara (sect. Dulcamara) were compared with each other and with the 5S rDNA of Lycopersicon esculentum. The length of the repeat ranges from 285 bp to 349 bp. The complete 5S repeat unit consists of the 120-bp long conserved coding region and of a intergenic spacer with a high variability in the central portion as result of deletions/duplications of short motifs demonstrating sequence similarity to box C in the 5S rRNA coding region. Numerous structural rearrangements found in the spacer region can be applied to design species-specific molecular markers for Solanum species involved in breeding programs. Characteristic insertions/deletions (indels) were used to reconstruct phylogenetic relationships among the species studied. S.dulcamara forms a separate clade; L. esculentum is more related to Solanum species of sect. Petota. Conservation of ancestral 5S spacer organization was demonstrated for the representatives of several series of sect. Petota, both Stellata and Rotata. Further rearrangements of the spacer organization occurred in at least four independent lineages: (1) L. esculentum, (2) ser. Polyadenia, (3) other Stellata species from Middle America (ser. Pinnatisecta and Bulbocastana), (4) superser. Rotata. In this last group, series Megistacroloba and Conocibaccata show a common origin, and separation from ser. Tuberosa.Solanum chacoense and Solanum maglia demonstrate a close relatedness to species of ser. Tuberosa and should be included into this group, whereas Solanum bukasovii should be excluded due to conservation of ancestral spacer organization. Three major subgroups may be distinguished for species from ser. Tuberosa, although a high sequence similarity was found here. Several wild species (diploids Solanum phureja and Solanum spegazzinii) probably participated in the natural origin of tetraploid S. tuberosum;others were later used for crossing in breeding programs (e.g. Solanum demissum). Clear separation of Middle-American Stellata species from South-American Stellata and from Middle-American Rotata polyploids is shown.

Molecular organization and evolution of the external transcribed rDNA spacer region in two diploid relatives ofNicotiana tabacum (Solanaceae)

For two close diploid relatives of the allotetraploidNicotiana tabacum, N. sylvestris (subg.Petunioides) andN. tomentosiformis (subg.Nicotiana), the nucleotide sequences of the external transcribed rDNA spacer regions (ETS) are described and compared (within the genus and with otherSolanaceae). The ETS of these two diploid species consists of three regions: the most upstream region I contains the putative RNA polymerase I transcription initiation site. Region II consists of several subrepeats (type A) and of a short species specific element. There are 5 subrepeats in theN. sylvestris clone and 10 subrepeats in the clone isolated fromN. tomentosiformis. Region III, adjacent to the 18S rRNA gene, exhibits much higher similarities between species than regions I and II, not only amongN. sylvestris andN. tomentosiformis but also with more distantly relatedSolanaceae. At the 5′-end of region III of the ETS in the twoNicotiana species studied in detail there are three copies of highly divergent subrepeats (type B). Possible ways of the step-wise molecular evolution of these ribosomal DNA regions inNicotiana and theSolanaceae are discussed.

Molecular evolution of 35S rDNA and taxonomic status of Lycopersicon within Solanum sect. Petota

To clarify the taxonomic status of tomatoes (“Lycopersicon”) and their relationship to the members of sect. Petota of genus Solanum L., organization of the rDNA external transcribed spacer (5′ ETS) was studied in 33 Solanum and “Lycopersicon” species. Phylogenetic reconstruction revealed that three major groups can be distinguished. Non-tuber-bearing species of ser. Etuberosa as well as tuber-bearing Central American diploids appeared as a paraphyletic group. The first of two well-defined clades embraced all tuber-bearing South American species and Central American polyploids. The other clade (named “tomato clade”) contains non-tuber-bearing species of ser. Juglandifolia and tomato species of ser. Neolycopersicon, which appears to be imbedded in sect. Petota. The new 5′ ETS variant D characterized by a cluster of downstream subrepeats is characteristic for the tomato clade. The variant D originated directly from the most ancestral variant A found in ser. Etuberosa and the Central American diploids, whereas variants B and C specific for the tuber-bearing South American species and Central American polyploids represent a parallel lineage of molecular evolution. The sequence analysis demonstrates the existence of an evolutionary trend of parallel multiplication of specific motifs in 5′ ETS in different groups of sect. Petota.