High-throughput microsatellite genotyping in ecology: improved accuracy, efficiency, standardization and success with low-quantity and degraded DNA.

Microsatellite markers have played a major role in ecological, evolutionary and conservation research during the past 20 years. However, technical constrains related to the use of capillary electrophoresis and a recent technological revolution that has impacted other marker types have brought to question the continued use of microsatellites for certain applications. We present a study for improving microsatellite genotyping in ecology using high-throughput sequencing (HTS). This approach entails selection of short markers suitable for HTS, sequencing PCR-amplified microsatellites on an Illumina platform and bioinformatic treatment of the sequence data to obtain multilocus genotypes. It takes advantage of the fact that HTS gives direct access to microsatellite sequences, allowing unambiguous allele identification and enabling automation of the genotyping process through bioinformatics. In addition, the massive parallel sequencing abilities expand the information content of single experimental runs far beyond capillary electrophoresis. We illustrated the method by genotyping brown bear samples amplified with a multiplex PCR of 13 new microsatellite markers and a sex marker. HTS of microsatellites provided accurate individual identification and parentage assignment and resulted in a significant improvement of genotyping success (84%) of faecal degraded DNA and costs reduction compared to capillary electrophoresis. The HTS approach holds vast potential for improving success, accuracy, efficiency and standardization of microsatellite genotyping in ecological and conservation applications, especially those that rely on profiling of low-quantity/quality DNA and on the construction of genetic databases. We discuss and give perspectives for the implementation of the method in the light of the challenges encountered in wildlife studies.

Structure, function, and evolution of ferritins.

The ferritins of animals and plants and the bacterioferritins (BFRs) have a common iron-storage function in spite of differences in cytological location and biosynthetic regulation. The plant ferritins and BFRs are more similar to the H chains of mammals than to mammalian L chains, with respect to primary structure and conservation of ferroxidase center residues. Hence they probably arose from a common H-type ancestor. The recent discovery in E. coli of a second type of iron-storage protein (FTN) resembling ferritin H chains raises the question of what the relative roles of these two proteins are in this organism. Mammalian L ferritins lack ferroxidase centers and form a distinct group. Comparison of the three-dimensional structures of mammalian and invertebrate ferritins, as well as computer modeling of plant ferritins and of BFR, indicate a well conserved molecular framework. The characterisation of numerous ferritin homopolymer variants has allowed the identification of some of the residues involved in iron uptake and an investigation of some of the functional differences between mammalian H and L chains.

Ferritin accumulation and degradation in different organs of pea (Pisum sativum) during development.

Iron concentration and ferritin distribution have been determined in different organs of pea (Pisum sativum) during development under conditions of continuous iron supply from hydroponic cultures. No ferritin was detected in total protein extracts from roots or leaves. However, a transient iron accumulation in the roots, which corresponds to an increase in iron uptake, was observed when young fruits started to develop. Ferritin was detectable in total protein extracts of flowers and pods, and it accumulated in seeds. In seeds, the same relative amount of ferritin was detected in cotyledons and in the embryo axis. In cotyledons, ferritin and iron concentration decrease progressively during the first week of germination. Ferritin in the embryo axis was processed, and disappeared, during germination, within the first 4 days of radicle and epicotyl growth. This degradation of ferritin in vivo was marked by a shortening of a 28 kDa subunit, giving 26.5 and 25 kDa polypeptides, reminiscent of the radical damage occurring in pea seed ferritin during iron exchange in vitro [Laulhere, Laboure & Briat (1989) J. Biol. Chem. 264, 3629-3635]. Developmental control of iron concentration and ferritin distribution in different organs of pea is discussed.

Structure and differential expression of the four members of the Arabidopsis thaliana ferritin gene family.

Four ferritin genes are found within the complete sequence of the Arabidopsis thaliana genome. All of them are expressed and their corresponding cDNA species have been cloned. The polypeptide sequences deduced from these four genes confirm all the properties of the ferritin subunits described so far, non-exhaustively, from various plant species. All are predicted to be targeted to the plastids, which is consistent with the existence of a putative transit peptide at their N-terminal extremity. They also all possess a conserved extension peptide in the mature subunit. Specific residues for ferroxidase activity and iron nucleation, which are found respectively in H-type or L-type ferritin subunits in animals, are both conserved within each of the four A. thaliana ferritin polypeptides. In addition, the hydrophilic nature of the plant ferritin E-helix is conserved in the four A. thaliana ferritin subunits. Besides this strong structural conservation, the four genes are differentially expressed in response to various environmental signals, and during the course of plant growth and development. AtFer1 and AtFer3 are the two major genes expressed in response to treatment with an iron overload. Under our experimental conditions, AtFer4 is expressed with different kinetics and AtFer2 is not responsive to iron. H(2)O(2) activates the expression of AtFer1 and, to a smaller extent, AtFer3. Abscisic acid promotes the expression of only AtFer2, which is consistent with the observation that this is the only gene of the four to be expressed in seeds, whereas AtFer1, AtFer4 and AtFer3 are expressed in various vegetative organs but not in seeds.

Inhibition of the iron-induced ZmFer1 maize ferritin gene expression by antioxidants and serine/threonine phosphatase inhibitors.

Two pathways have been implicated in the regulation of maize ferritin synthesis in response to iron. One of them involves the plant hormone abscisic acid (ABA) and controls the expression of ZmFer2 gene(s). Another pathway, ABA-independent, has been characterized in a de-rooted maize plantlet system and involves an oxidative step. The ZmFer1 maize ferritin gene is not regulated by ABA, and it is shown in this paper that the corresponding mRNA accumulates in de-rooted maize plantlets and BMS (Black Mexican Sweet) maize cell suspension cultures in response to iron via the oxidative pathway described previously. To investigate ZmFer1 gene regulation further, the BMS cell system has been used to develop a transient expression assay using a ZmFer1-beta-glucuronidase fusion. Both iron induction and antioxidant inhibition of ZmFer1 gene expression were observed in this system. Using Northern blot analysis and transient expression experiments, it was shown that both okadaic acid and calyculin A, two serine/ threonine phosphatase inhibitors, specifically inhibit ZmFer1 gene expression. These data indicate that an okadaic acid-sensitive protein phosphatase activity is involved in the regulation of the ZmFer1 ferritin gene in maize cells, and this activity is required for iron-induced expression of this gene.

Iron induces ferritin synthesis in maize plantlets.

The iron-storage protein ferritin has been purified to homogeneity from maize seeds, allowing to determine the sequence of the first 29 NH2-terminal amino acids of its subunit and to raise specific rabbit polyclonal antibodies. Addition of 500 microM Fe-EDTA/75 microM Fe-citrate to hydroponic culture solutions of maize plantlets, previously starved for iron, led to a significant increase of the iron concentration of roots and leaves, albeit root iron was mainly found associated with the apoplast. Immunodetection of ferritin by western blots indicated that this iron treatment induced ferritin protein accumulation in roots and leaves over a period of 3 days. In order to investigate this induction at the ferritin mRNA level, various ferritin cDNA clones were isolated from a cDNA library prepared from poly(A)+ mRNA isolated from roots 48 h after iron treatment. These cDNAs were classified into two groups called FM1 and FM2. Upstream of the sequence encoding the mature ferritin subunit, both of these cDNAs contained an in-frame coding sequence with the characteristics of a transit peptide for plastid targeting. Two members of the FM1 subfamily, both partial at their 5' extremity, were characterized. They are identical, except in their 3' untranslated region: FM1A extends 162 nucleotides beyond the 3' terminus of FM1B. These two mRNAs could arise from the use of two different polyadenylation signals. FM2 is 96% identical to FM1 and contains 45 nucleotides of 5' untranslated region. Northern analyses of root and leaf RNAs, at different times after iron treatment, revealed ferritin mRNA accumulation in response to iron. Ferritin mRNA accumulation was transient and particularly abundant in leaves, reaching a maximum at 24 h. The level of ferritin mRNA in roots was affected to a lesser extent than in leaves.

Abscisic acid is involved in the iron-induced synthesis of maize ferritin.

The ubiquitous iron storage protein ferritin has a highly conserved structure in plants and animals, but a distinct cytological location and a different level of control in response to iron excess. Plant ferritins are plastid-localized and transcriptionally regulated in response to iron, while animal ferritins are found in the cytoplasm and have their expression mainly controlled at the translational level. In order to understand the basis of these differences, we developed hydroponic cultures of maize plantlets which allowed an increase in the intracellular iron concentration, leading to a transient accumulation of ferritin mRNA and protein (Lobréaux,S., Massenet,O. and Briat,J.F., 1992, Plant Mol. Biol., 19, 563-575). Here, it is shown that iron induces ferritin and RAB (Responsive to Abscisic Acid) mRNA accumulation relatively with abscisic acid (ABA) accumulation. Ferritin mRNA also accumulates in response to exogenous ABA. Synergistic experiments demonstrate that the ABA and iron responses are linked, although full expression of the ferritin genes cannot be entirely explained by an increase in ABA concentration. Inducibility of ferritin mRNA accumulation by iron is dramatically decreased in the maize ABA-deficient mutant vp2 and can be rescued by addition of exogenous ABA, confirming the involvement of ABA in the iron response in plants. Therefore, it is concluded that a major part of the iron-induced biosynthesis of ferritin is achieved through a pathway involving an increase in the level of the plant hormone ABA. The general conclusion of this work is that the synthesis of the same protein in response to the same environmental signal can be controlled by separate and distinct mechanisms in plants and animals.

Regulation of plant ferritin synthesis: how and why.

Plant ferritins are key iron-storage proteins that share important structural and functional similarities with animal ferritins. However, specific features characterize plant ferritins, among which are plastid cellular localization and transcriptional regulation by iron. Ferritin synthesis is developmentally and environmentally controlled, in part through the differential expression of the various members of a small gene family. Furthermore, a strict requirement for plant ferritin synthesis regulation is attested to by alterations of the photosynthetic apparatus and of iron homeostasis in transgenic tobaccos overexpressing these proteins. Plant ferritin gene regulation appears to consist of a complex interplay of transcriptional and posttranscriptional mechanisms, involving cellular relays such as plant hormones, oxidative steps and Ser/Thr phosphatase.

Amino-acid sequence and predicted three-dimensional structure of pea seed (Pisum sativum) ferritin.

The iron storage protein, ferritin, is widely distributed in the living kingdom. Here the complete cDNA and derived amino-acid sequence of pea seed ferritin are described, together with its predicted secondary structure, namely a four-helix-bundle fold similar to those of mammalian ferritins, with a fifth short helix at the C-terminus. An N-terminal extension of 71 residues contains a transit peptide (first 47 residues) responsible for plastid targetting as in other plant ferritins, and this is cleaved before assembly. The second part of the extension (24 residues) belongs to the mature subunit; it is cleaved during germination. The amino-acid sequence of pea seed ferritin is aligned with those of other ferritins (49% amino-acid identity with H-chains and 40% with L-chains of human liver ferritin in the aligned region). A three-dimensional model has been constructed by fitting the aligned sequence to the coordinates of human H-chains, with appropriate modifications. A folded conformation with an 11-residue helix is predicted for the N-terminal extension. As in mammalian ferritins, 24 subunits assemble into a hollow shell. In pea seed ferritin, its N-terminal extension is exposed on the outside surface of the shell. Within each pea subunit is a ferroxidase centre resembling those of human ferritin H-chains except for a replacement of Glu-62 by His. The channel at the 4-fold-symmetry axes defined by E-helices, is predicted to be hydrophilic in plant ferritins, whereas it is hydrophobic in mammalian ferritins.

Structure and differential expression of two maize ferritin genes in response to iron and abscisic acid.

In plants, synthesis of the iron-storage protein ferritin in response to iron is not regulated at the translational level; this is in contrast to ferritin synthesis in animals. Part of the response is mediated through a transduction pathway which involves the plant hormone abscisic acid. In this work, we report the cloning and sequencing of two maize ferritin genes (ZmFer1 and ZmFer2) coding for members of the two ferritin mRNA subclasses, FM1 and FM2, respectively. Although plant and animal ferritins are closely related proteins, a major difference is observed between the organisation of the genes. Both maize ferritin genes are organised as eight exons and seven introns, the positions of which are identical within the two genes, while animal ferritin genes are interrupted by three introns, at positions different from those found in maize genes. Sequence divergence between the 3' untranslated regions of these genes has allowed the use of specific probes to study the accumulation of FM1 and FM2 transcripts in response to various environmental cues. Such probes have shown that FM1 and FM2 transcripts accumulate with differential kinetics in response to iron; FM1 mRNA accumulate earlier than FM2 mRNA and only FM2 transcripts accumulate in response to exogenous abscisic acid or water stress. Mapping of the transcriptional initiation region of these two genes defined their 5' upstream regions and allowed a sequence comparison of their promoters, which appeared highly divergent. This raises the possibility that the differential accumulation of FM1 and FM2 mRNAs in response to iron, abscisic acid and drought could be due to differential transcription of ZmFer1 and ZmFer2.

Nickel resistance and chromatin condensation in Saccharomyces cerevisiae expressing a maize high mobility group I/Y protein.

Expression of a maize cDNA encoding a high mobility group (HMG) I/Y protein enables growth of transformed yeast on a medium containing toxic nickel concentrations. No difference in the nickel content was measured between yeast cells expressing either the empty vector or the ZmHMG I/Y2 cDNA. The ZmHMG I/Y2 protein contains four AT hook motifs known to be involved in binding to the minor groove of AT-rich DNA regions. HMG I/Y proteins may act as architectural elements modifying chromatin structure. Indeed, a ZmHMG I/Y2-green fluorescent protein fusion protein was observed in yeast nuclei. Nickel toxicity has been suggested to occur through an epigenetic mechanism related to chromatin condensation and DNA methylation, leading to the silencing of neighboring genes. Therefore, the ZmHMG I/Y2 protein could prevent nickel toxicity by interfering with chromatin structure. Yeast cell growth in the presence of nickel and yeast cells expressing the ZmHMG I/Y2 cDNA increased telomeric URA3 gene silencing. Furthermore, ZmHMG I/Y2 restored a wild-type level of nickel sensitivity to the yeast (Delta)rpd3 mutant. Therefore, nickel resistance of yeast cells expressing the ZmHMG I/Y2 cDNA is likely achieved by chromatin structure modification, restricting nickel accessibility to DNA.

Characterization of an iron-dependent regulatory sequence involved in the transcriptional control of AtFer1 and ZmFer1 plant ferritin genes by iron.

In eukaryotic cells, ferritin synthesis is controlled by the intracellular iron status. In mammalian cells, iron derepresses ferritin mRNA translation, whereas it induces ferritin gene transcription in plants. Promoter deletion and site-directed mutagenesis analysis, combined with gel shift assays, has allowed identification of a new cis-regulatory element in the promoter region of the ZmFer1 maize ferritin gene. This Iron-Dependent Regulatory Sequence (IDRS) is responsible for transcriptional repression of ZmFer1 under low iron supply conditions. The IDRS is specific to the ZmFer1 iron-dependent regulation and does not mediate the antioxidant response that we have previously reported (Savino et al. (1997) J. Biol. Chem. 272, 33319-33326). In addition, we have cloned AtFer1, the Arabidopsis thaliana ZmFer1 orthologue. The IDRS element is conserved in the AtFer1 promoter region and is functional as shown by transient assay in A. thaliana cells and stable transformation in A. thaliana transgenic plants, demonstrating its ubiquity in the plant kingdom.

Nuclear import in permeabilized protoplasts from higher plants has unique features.

The import of proteins into the nucleus is a poorly understood process that is thought to require soluble cytosolic factors in vertebrates and yeast. To test this model in plants and to identify components of the import apparatus, we developed a direct in vitro nuclear import assay by using tobacco protoplasts that were permeabilized without detergents such as digitonin or Triton X-100. Substrates were imported specifically by a mechanism that required only guanine nucleotides. Moreover, in vitro import did not require exogenous cytosol. To investigate this novel finding, we isolated a full-length cDNA encoding an Arabidopsis homolog of vertebrate and yeast nuclear localization signal receptors and produced an affinity-purified antibody. The plant receptor was tightly associated with cellular components in permeabilized protoplasts, even in the presence of 0.1% Triton X-100, indicating that this factor and probably others were retained to an extent sufficient to support import. The lectin wheat germ agglutinin bound to the nucleus; however, it did not block translocation in our system, indicating that direct interaction with polysaccharide modifications at the nuclear pore complex was probably not essential for import in plants. Other features of in vitro import included reduced but significant import at low temperature.