An En/Spm based transposable element system for gene isolation in Arabidopsis thaliana

At the start of the research described in this thesis, the main aim was to develop, study and apply an efficient En/Spm-I/dSpm based transposon tagging system in Arabidopsis thaliana to generate tagged mutants and to provide insights in the possibilities for future applications of such a transposon tagging system in studying plant gene functions. The first step was the introduction of an active En/Spm-I/dSpm system into Arabidopsis. Initially a very simple T-DNA construct was transformed, containing a nearly full En-1 element, without left and right border sequences, and with its promoter replaced by the stronger CaMV 35S promoter. As the same construct harboured a non-autonomous I/dSpm element, only one T-DNA transformation was needed. Transformation of this 'in cis two- element En/Spm-I/dSpm system' yielded one transformant with two T-DNA insertion loci, TEn2 and TEn5, each having one, respectively, five 35S -En/Spm t ransposase gene copies (Chapter 1). The transposition activity of the I/dSpm elements turned out to be surprisingly high. Instead of a germinal excision frequency, which was generally used to express the activity of heterologous transposable element systems, the term independent transposition frequency or itf was coined, as a measure accounting for the entire transposition process. Although not always easy to compare for different transposable element systems, an average itf of over 10%, as was found for this En/Spm-I/dSpm system (Chapter 1), has not been reported for any of the Ac-Ds based heterologous transposon tagging systems developed for Arabidopsis (Bancroft et al., 1992; Bhatt et al., 1996; Fedoroff and Smith, 1993; Honma et al., 1993; Long et al., 1993b; Swinburne et al., 1992). Obtaining a high transposition frequency in Ac-Ds systems is hampered by the fact that the transposase acts as an inhibitor of Ac or Ds transposition when its expression exceeds a certain level. Apparently such autoregulatory mechanism is not present in the 'in cis two-element En/Spm-I/dSpm system'.The need for a more sophisticated system diminished with the availability of this simple, but efficient En/Spm-I/dSpm transposon system and it was therefore studied in more detail to determine: 1) the ability to transpose continuously even after many plant generations; 2) the distribution of elements after transposition; 3) the ability to transpose to transcribed regions and 4) the ability to cause mutations. To start with the first issue, transposition has been studied in up to 12 generations, starting from the primary transformant. In all these generations, there was no apparent reduction in itf, demonstrating a continuous transposition of I/dSpm elements, irrespective of the generation number. The second issue, the distribution of elements after transposition, is another important aspect of a transposable element system. For maize transposons it is reported that the insertion site is preferentially physically and often genetically linked to the excision site (Dooner and Belachew, 1989; Peterson, 1970). This is not so remarkable considering that the proteins that perform the transposition steps will have a higher chance of encountering a nearby site on the genome instead of a distant DNA sequence. Like in maize (Peterson, 1970), the I/dSpm elements show a preference for insertion in genetically linked sites (Chapter 1), but the preference is not very explicit. Based on the observations of transpositions from tagged genes to sites within a few cM or only several kb away, and on the analogy to the En/Spm-I/dSpm elements in maize, the overall estimate is that about 30% of the elements transpose to sites genetically linked to the excision site. The mapped elements (Chapter 5) show a fairly even distribution over the genome, although there seems to be some clustering of elements (from different origin) to certain genomic regions, like the top of chromosome 4, the bottom of chromosome 1 and the lower half of chromosome 5.In accordance with the idea that DNA must be in an open confirmation to allow the access of transposase proteins before transposition (Zhang and Spradling, 1993), there are many indications that I/dSpm elements insert in regions of the Arabidopsis genome containing genes: a) I/dSpm flanking DNAs rarely contain repetitive sequences, but are mostly single copy sequences, as are genes (Chapters 1 and 5); b) about half of the I/dSpm elements are inserted in relatively conserved genomic regions, with no RFLPs for five restriction enzymes (Chapters 1 and 5); c) at least one third of the examined I/dSpm elements is inserted in close vicinity of transcriptionally active genomic sequences (Chapter 7). Insertion in unique, conserved and often transcribed DNA may not seem surprising for a plant species with little repetitive DNA and a small genome with a high gene density (Meyerowitz, 1989). However, a high frequency of insertion into genic regions of the genome offers the best chances for gene tagging.The most important aspect of a transposable element system is the possibility to generate tagged mutants. The En/Spm-I/dSpm system is mutagenic, with as much as 12 tagged mutants found so far. Most of these were obtained after screening for random mutant phenotypes. When screening for specific mutants, such as reduced seed dormancy, tagged alleles of the ABI3 and LEC1 genes were found (M. Koornneef et al., unpublished results). Mutants at the CER6 locus were obtained by targeted tagging, using the nearby ap1::I/dSpm allele as the I/dSpm element donor (A. Pereira, unpublished results). These selected and targeted transposon tagging experiments are illustrative examples of the feasibility to efficiently isolate tagged mutants of a special phenotypic or genotypic class.Ideally, a population saturated with different I/dSpm insertions can be made, allowing the isolation of mutants for virtually every gene. Such a 'mutation machine' can be further used for PCR based targeted gene inactivation. This novel technique, which was originally developed for Drosophila melanogaster (see O'Hare, 1990), exploits the abundance of transposons for the identification of insertions in genes with known DNA sequence, but no known mutant phenotype. In general, DNAs from multidimensional pools of individuals from a large population are used for a PCR using two primers. One primer is specific for the transposon terminus (directed outwards), the other is specific for the target gene. A fragment can only be amplified when a transposon insert is close enough to the target primer. This technique has been shown to work in Drosophila (Ballinger and Benzer, 1989; Kaiser and Goodwin, 1990), Caenorhabditis elegans (Zwaal et al., 1993), Petunia hybrida (Koes. et al., 1995) and maize (Das and Martienssen, 1995), using 'mutation machine' transposable element systems.Two I/dSpm tagged genes that have been studied in great detail are the MS2 gene (Chapters 2 and 3) and the CER1 gene (Chapter 4). Mutants for both genes display a conditional male sterile phenotype, which is the only mutant phenotype for the ms2 mutant, but for the cer1 mutant it is a pleiotropic effect of a deficiency in epicuticular wax biosynthesis. The ms2 mutants are occasionally able to self-fertilize, especially in high relative humidity and late in plant development, but seed set rarely reaches more than a few percentages of wild-type seed set. This in contrast to the cer1 mutants, which are male sterile in low relative humidity (≤50% RH) and fertile in high relative humidity (≥95% RH). Fertility cannot be completely restored by environment in the ms2 mutants due to the drastic effect of the mutation on pollen development. The MS2 gene is expressed in the tapetum around the time of microspore release from the microspore mother cells. The gene is needed for the development of a proper exine layer, protecting the microspore from harmful environmental influences. Consequently the few ms2 microspores that are produced have very feeble pollen walls, which leaves only very few pollen grains intact for fertilization.The CER1 gene acts much later in pollen development. Phenotypically cer1 and wild-type pollen cannot be distinguished, apart from a difference in gerniination ability (Chapter 4). As cer1 pollen germination is like wild type when applying CER1 pollen or by pollinating in high relative humidity, there appears to be a substance missing from the pollen coat that is required for the necessary rehydration of a pollen grain. Although essential under low relative humidity conditions, this is only a minor defect, which can be easily overcome.Besides the similarity in mutant phenotypes, the MS2 gene and the CER1 gene share the characteristic of encoding proteins with homology to enzymes in the fatty acid biosynthesis pathway. The MS2 protein has most resemblance with the wax fatty acid reductase protein from the desert shrub jojoba, which is involved in the conversion of wax fatty acids to wax alcohols (Chapter 3). The CER1 protein shares structural features of fatty acid desaturases, and it has a proposed function as a decarbonylase, converting long carbon chain aldehydes to alkanes (Chapter 4). There are more examples of a correlation between male fertility and wax biosynthesis, as also other cer mutants such as cer3, 6, 8 and 10 are known to be disturbed in male fertility. It demonstrates the general importance of fatty acid biosynthesis for male gametogenesis.The last part of this thesis has been devoted to further applications of the En/Spm-I/dSpm tagging system in Arabidopsis for the analysis of plant gene functions. The first description of transposable elements as controlling elements (McClintock, 1948), was based on the effect transposons had on the expression of maize genes. Especially (d)Spm insertions were known to cause Spm dependent or suppressible gene expression (Fedoroff, 1989). This effect is now also described for the En/Spm- I/dSpm system in Arabidopsis (Chapter 6). As in maize, an En/Spm suppressible allele contains an anti-parallel I/dSpm element insertion, which can be spliced from the mRNA. This knowledge can be further used to design an artificial gene expression system, in which an introduced gene containing an anti-parallel I/dSpm element, can be negatively controlled in the presence of an En/Spm transposase source. The reverse effect, En/Spm dependent gene expression, is now also described for Arabidopsis (Chapter 6), but the mechanism for dependence is not yet understood. The availability of the En/Spm dependent lad::I/dSpm mutant will be useful for further research.A more general way to study gene expression is the use of gene traps as detectors of gene activity. The pilot experiments described in chapter 7, have shown the possibility to adapt I/dSpm elements as gene traps. Especially their advantage in detecting genes without the need for mutation, and the possibility of studying the activity of genes which are lethal as homozygous mutants, are important additional properties of gene trap systems over "traditional" transposon tagging. In combination with its efficient transposition behaviour, an En/Spm-I/dSpm based gene trap tagging system seems an attractive alternative for the existing Ac-Ds or T-DNA based gene trap systems.Summarizing, an En/Spm-I/dSpm transposon tagging system has been well developed for Arabidopsis and many of its basic characteristics are studied and described. I/dSpm tagged mutants can be found with reasonable frequencies, either by random, selected or targeted tagging strategies. The cloning and characterization of two genes affecting male fertility has been described. Further ways to improve tagging frequencies, based on phenotypic or on genotypic selection have been discussed. In addition, the system can be exploited to study plant gene expression and gene function either by En/Spm controlled activity of I/dSpm tagged genes, or by using I/dSpm gene detector elements

Mapping QTLs for mineral accumulation and shoot dry biomass under different Zn nutritional conditions in Chinese cabbage ( Brassica rapa L. ssp. pekinensis )

Abstract Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the most important vegetables in China. Genetic dissection of leaf mineral accumulation and tolerance to Zn stress is important for the improvement of the nutritional quality of Chinese cabbage by breeding. A mapping population with 183 doubled haploid (DH) lines was used to study the genetics of mineral accumulation and the growth response to Zn. The genetic map was constructed based on 203 AFLPs, 58 SSRs, 22 SRAPs and four ESTPs. The concentration of 11 minerals was determined in leaves for 142 DH lines grown in an open field. In addition shoot dry biomass (SDB) under normal, deficient and excessive Zn nutritional conditions were investigated in hydroponics experiments. Ten QTLs, each explaining 11.1¿17.1% of the Na, Mg, P, Al, Fe, Mn, Zn and Sr concentration variance, were identified by multiple-QTL model (MQM) mapping. One common QTL was found affecting SDB under normal, deficient and excessive Zn nutritional conditions. An additional QTL was detected for SDB under Zn excess stress only. These results offer insights into the genetic basis of leaf mineral accumulation and plant growth under Zn stress conditions in Chinese cabbag

Identification and functional analysis of two ZIP metal transporters of the hyperaccumulator Thlaspi caerulescens

Abstract The heavy metal hyperaccumulator Thlaspi caerulescens expresses several ZIP-like genes at higher levels than their orthologues in non-hyperaccumulator species, but it is not clear why. To elucidate the function of the T. caerulescens orthologues of the Arabidopsis thaliana ZIP5 and ZIP6 genes, full-length cDNAs of TcZNT5-LC and TcZNT6-LC were cloned, their expression was examined and genes were expressed in A. thaliana. Transcript level analysis revealed the constitutively high expression of these two genes in T. caerulescens compared to AtZIP5 and AtZIP6 genes and differential expression of both genes when comparing two accessions of T. caerulescens with different metal accumulation properties. Expression of TcZNT5-LC in A. thaliana did not modify Cd or Zn tolerance, but mildly affected the root and shoot Zn concentrations towards a hyperaccumulator shoot to root concentration ratio. A. thaliana zip5 knock-out mutants showed increased tolerance to Cd and decreased seed mineral concentrations. Expression of TcZNT6-LC enhanced the Cd sensitivity of A. thaliana, but no phenotype was observed for the zip6 mutant. In conclusion, the changes in expression of TcZNT5-LC and TcZNT6-LC upon changes in Zn or Cd exposure indicate both genes act in metal homeostasis, but their CaMV 35S-mediated expression in A. thaliana does not create T. caerulescens hyperaccumulator phenotype

Gomphrena claussenii, the first South-American metallophyte species with indicator-like Zn and Cd accumulation and extreme metal tolerance

Plant species with the capacity to tolerate heavy metals are potentially useful for phytoremediation since they have adapted to survive and reproduce under toxic conditions and to accumulate high metal concentrations. Gomphrena claussenii Moq., a South-American species belonging to the Amaranthaceae, is found at a zinc (Zn) mining area in the state of Minas Gerais, Brazil. Through soil and hydroponic experiments, the metal tolerance and accumulation capacities of G. claussenii were assessed and the effects on physiological characteristics were compared with a closely related non-tolerant species, G. elegans Mart. G. claussenii plants grown in soil sampled at the Zn smelting area accumulated up to 5318µgg-1 of Zn and 287 µg g-1 of cadmium (Cd) in shoot dry biomass after 30 days of exposure. Plants were grown in hydroponics containing up to 3000 µM of Zn and 100 µM of Cd for G. claussenii and 100 µM of Zn and 5 µM of Cd for G. elegans. G. claussenii proved to be an extremely tolerant species to both Zn and Cd, showing only slight metal toxicity symptoms at the highest treatment levels, without significant decrease in biomass and no effects on root growth, whereas the non-tolerant species G. elegans showed significant toxicity effects at the highest exposure levels. Both species accumulated more Zn and Cd in roots than in shoots. In G. elegans, over 90% of the Cd remained in the roots, but G. claussenii showed a root:shoot concentration ratio of around 2, with shoots reaching 0.93% Zn and 0.13% Cd on dry matter base. In G. claussenii shoots, the concentrations of other minerals, such as iron (Fe) and manganese (Mn), were only affected by the highest Zn treatment while in G. elegans the Fe and Mn concentrations in shoots decreased drastically at both Zn and Cd treatments. Taking together, these results indicate that G. claussenii is a novel metallophyte, extremely tolerant of high Zn and Cd exposure and an interesting species for further phytoremediation studies Keywords: phytoremediation, Zn/Cd hypertolerance, hyperaccumulation, metal contamination, Gomphrena claussenii, Gomphrena elegan

Comparative transcriptome analysis of the metal hyperaccumulator Noccaea caerulescens

The metal hyperaccumulator Noccaea caerulescens is an established model to study the adaptation of plants to metalliferous soils. Various comparators have been used in these studies. The choice of suitable comparators is important and depends on the hypothesis to be tested and methods to be used. In high-throughput analyses such as microarray, N. caerulescens has been compared to non-tolerant, non-accumulator plants like Arabidopsis thaliana or Thlaspi arvense rather than to the related hypertolerant or hyperaccumulator plants. An underutilized source is N. caerulescens populations with considerable variation in their capacity to accumulate and tolerate metals. Whole transcriptome sequencing (RNA-Seq) is revealing interesting variation in their gene expression profiles. Combining physiological characteristics of N. caerulescens accessions with their RNA-Seq has a great potential to provide detailed insight into the underlying molecular mechanisms, including entirely new gene products. In this review we will critically consider comparative transcriptome analyses carried out to explore metal hyperaccumulation and hypertolerance of N. caerulescens, and demonstrate the potential of RNA-Seq analysis as a tool in evolutionary genomic

A comprehensive set of transcript sequences of the heavy metal hyperaccumulator Noccaea caerulescens

Noccaea caerulescens is an extremophile plant species belonging to the Brassicaceae family. It has adapted to grow on soils containing high, normally toxic, concentrations of metals such as nickel, zinc, and cadmium. Next to being extremely tolerant to these metals, it is one of the few species known to hyperaccumulate these metals to extremely high concentrations in their aboveground biomass. In order to provide additional molecular resources for this model metal hyperaccumulator species to study and understand the mechanism of adaptation to heavy metal exposure, we aimed to provide a comprehensive database of transcript sequences for N. caerulescens. In this study, 23,830 transcript sequences (isotigs) with an average length of 1025 bp were determined for roots, shoots and inflorescences of N. caerulescens accession “Ganges” by Roche GS-FLEX 454 pyrosequencing. These isotigs were grouped into 20,378 isogroups, representing potential genes. This is a large expansion of the existing N. caerulescens transcriptome set consisting of 3705 unigenes. When translated and compared to a Brassicaceae proteome set, 22,232 (93.2%) of the N. caerulescens isotigs (corresponding to 19,191 isogroups) had a significant match and could be annotated accordingly. Of the remaining sequences, 98 isotigs resembled non-plant sequences and 1386 had no significant similarity to any sequence in the GenBank database. Among the annotated set there were many isotigs with similarity to metal homeostasis genes or genes for glucosinolate biosynthesis. Only for transcripts similar to Metallothionein3 (MT3), clear evidence for an additional copy was found. This comprehensive set of transcripts is expected to further contribute to the discovery of mechanisms used by N. caerulescens to adapt to heavy metal exposur

Identification of seed-related QTL in Brassica rapa

To reveal the genetic variation, and loci involved, for a range of seed-related traits, a new F2 mapping population was developed by crossing Brassica rapa ssp. parachinensis L58 (CaiXin) with B. rapa ssp. trilocularis R-o-18 (spring oil seed), both rapid flowering and self-compatible. A linkage map was constructed using 97 AFLPs and 21 SSRs, covering a map distance of 757 cM with an average resolution of 6.4 cM, and 13 quantitative trait loci (QTL) were detected for nine traits. A strong seed colour QTL (LOD 26) co-localized with QTL for seed size (LOD 7), seed weight (LOD 4.6), seed oil content (LOD 6.6), number of siliques (LOD 3) and number of seeds per silique (LOD 3). There was only a significant positive correlation between seed colour and seed oil content in the yellow coloured classes. Seed coat colour and seed size were controlled by the maternal plant genotype. Plants with more siliques tended to have more, but smaller, seeds and higher seed oil content. Seed colour and seed oil content appeared to be controlled by two closely linked loci in repulsion phase. Thus, it may not always be advantageous to select for yellow-seededness when breeding for high seed oil content in Brassicas

Isotopic signatures reveal zinc cycling in the natural habitat of hyperaccumulator Dichapetalum gelonioides subspecies from Malaysian Borneo

Background: Some subspecies of Dichapetalum gelonioides are the only tropical woody zinc (Zn)-hyperaccumulator plants described so far and the first Zn hyperaccumulators identified to occur exclusively on non-Zn enriched 'normal' soils. The aim of this study was to investigate Zn cycling in the parent rock-soil-plant interface in the native habitats of hyperaccumulating Dichapetalum gelonioides subspecies (subsp. pilosum and subsp. sumatranum). We measured the Zn isotope ratios (δ66Zn) of Dichapetalum plant material, and associated soil and parent rock materials collected from Sabah (Malaysian Borneo). Results: We found enrichment in heavy Zn isotopes in the topsoil (δ66Zn 0.13 ‰) relative to deep soil (δ66Zn -0.15 ‰) and bedrock (δ66Zn -0.90 ‰). This finding suggests that both weathering and organic matter influenced the Zn isotope pattern in the soil-plant system, with leaf litter cycling contributing significantly to enriched heavier Zn in topsoil. Within the plant, the roots were enriched in heavy Zn isotopes (δ66Zn ~ 0.60 ‰) compared to mature leaves (δ66Zn ~ 0.30 ‰), which suggests highly expressed membrane transporters in these Dichapetalum subspecies preferentially transporting lighter Zn isotopes during root-to-shoot translocation. The shoots, mature leaves and phloem tissues were enriched in heavy Zn isotopes (δ66Zn 0.34–0.70 ‰) relative to young leaves (δ66Zn 0.25 ‰). Thisindicates that phloem sources are enriched in heavy Zn isotopes relative to phloem sinks, likely because of apoplastic retention and compartmentalization in the Dichapetalum subspecies. Conclusions: The findings of this study reveal Zn cycling in the rock-soil-plant continuum within the natural habitat of Zn hyperaccumulating subspecies of Dichapetalum gelonioides from Malaysian Borneo. This study broadens our understanding of the role of a tropical woody Zn hyperaccumulator plant in local Zn cycling, and highlights the important role of leaf litter recycling in the topsoil Zn budget. Within the plant, phloem plays key role in Zn accumulation and redistribution during growth and development. This study provides an improved understanding of the fate and behaviour of Zn in hyperaccumulator soil-plant systems, and these insights may be applied in the biofortification of crops with Zn.Antony van der Ent, Philip Nti Nkrumah, Mark G. M. Aarts, Alan J. M. Baker, Fien Degryse, Chris Wawryk, and Jason K. Kirb