Development of retrotransposon-based markers IRAP and REMAP for cassava (Manihot esculenta).

Retrotransposons are abundant in the genomes of plants. In the present study, inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers were developed for the cassava genome (Manihot esculenta Crantz). Four cassava cultivars (Fécula Branca, IPR-União, Olho Junto, and Tamboara, two samples per cultivar) were used to obtain IRAP and REMAP fingerprints. Twelve designed primers were amplified alone and in combinations. The 42 IRAP/REMAP primer combinations amplified 431 DNA segments (bands; markers) of which 36 (8.36%) were polymorphic. The largest number of informative markers (16) was detected using the primers AYF2 and AYF2xAYF4. The number of bands for each primer varied from 3 to 16, with an average of 10.26 amplified segments per primer. The size of the amplified products ranged between 100 and 7000 bp. The AYF2 primer generated the highest number of amplified segments and showed the highest number of polymorphic bands (68.75%). Two samples of each cassava cultivar were used to illustrate the usefulness and the polymorphism of IRAP/REMAP markers. IRAP and REMAP markers produced a high number of reproducible bands, and might be informative and reliable for investigation of genetic diversity and relationships among cassava cultivars.

MASISH: a database for gene expression in maize seeds.

UNLABELLED: Grass seeds are complex organs composed by multiple tissues and cell types that develop coordinately to produce a viable embryo. The identification of genes involved in seed development is of great interest, but systematic spatial analyses of gene expression on maize seeds at the cell level have not yet been performed. MASISH is an online database holding information for gene expression spatial patterns in maize seeds based on in situ hybridization experiments. The web-based query interface allows the execution of gene queries and provides hybridization images, published references and information of the analyzed genes. AVAILABILITY: http://masish.uab.cat/.

Molecular analysis of a putative transposable retroelement from the Zea genus with internal clusters of tandem repeats.

The molecular characterization of a recently discovered family of long repetitive sequences, termed ZLRS, is described. These elements belong to the class of moderate dispersed repetitive DNA and are specific to the Zea genus. An 8089-bp sequence from a Zea diploperennis ZLRS element have been elucidated. Sequence analysis reveals the presence of a long terminal repeat-like region, two clusters of different tandem repeats and several ORFs. On these grounds, ZLRS could be considered a new member of the superfamily of transposable retroelements. Tandems are present in the majority of ZLRS elements, they show an important stem-loop secondary structure predicted by the computer and their sequence conservation suggests a functional role.

Discovery of a Zdel transposable element in Zea species as a consequence of a retrotransposon insertion.

Nucleotide sequences similar to del1 retrotransposon from Lilium henryi have been discovered in Zea diploperennis as a consequence of finding a Zea retrotransposon element inserted into one of them. These sequences named Zdel (Zea del1-like) elements are present in all the Zea species (about 100 copies per haploid genome) and in Tripsacum dactyloides and absent from closely related genera. Sequences corresponding to gag and protease domains from a Zdel element have been identified. The Zdel protease sequence shows a conserved active site motif (DT/SG) from aspartic proteases. The high level of DNA methylation found in Zdel elements may be related to the observed absence of transcriptional activity.

Quantitative subproteomic analysis of germinating related changes in the scutellum oil bodies of Zea mays.

Oil bodies (OBs) were purified from the scutellum of mature maize embryos and from embryos 2 days after imbibition and their associated proteins were extracted and separated by 2-DE. Eighteen proteins were shown to be differentially accumulated, thirteen showed a higher accumulation in mature scutellum and five were highly accumulated in the germinating scutellum. Proteins were identified using LC-MS/MS. Besides previously known oil body protein oleosin, other proteins were identified in this study. Among accumulated proteins during imbibition are prohibitin 2, stress-inducible membrane pore protein Tim17 and manganese superoxide dismutase. Among the proteins whose amount decreases during imbibition are cupin 2, two different protein disulfide isomerases, a triosephosphate isomerase, a class IV heat shock protein, the embryonic protein DC-8, the 60S ribosomal protein P0, a nucleoside-diphosphate kinase, and a rubber elongation factor protein. Some of the identified proteins were previously located in organelles other than oil bodies, suggesting that OBs may interact with these organelles. We also suggest that OBs may act as transient storage depots for proteins that are temporally in excess.

A maize defective-kernel mutant (longcell) characterized by tubular cells, severe morphological alterations and induction of cell death.

Seeds of the longcell mutant in maize (Zea mays L) have a defective-kernel phenotype: the embryo aborts at the early coleoptilar stage and the endosperm is reduced in size. Mutant embryos have severe alterations in morphogenesis. They have a suspensor-, an embryo axis- and a scutellum-like structure, but the shoot apical meristem (SAM) is not formed. Scanning electron microscopy showed that most of the cells in longcell embryos are tubular and abnormally enlarged. The level of expression of several genes involved in basic metabolism is not severely affected during early and mid embryogenesis, but storage molecule accumulation is reduced. Genes which in normal conditions are only expressed after germination, are expressed during kernel development in the longcell seeds. Mutant embryos undergo cell death in late embryogenesis. Nuclei in dying embryos are TUNEL positive, and different genes coding for hydrolytic enzymes are up-regulated. The expression of genes related to oxidative stress is also altered in longcell embryos. These results lead us to suggest that the longcell mutant may be cytokinesis-defective.

Envelope-class retrovirus-like elements are widespread, transcribed and spliced, and insertionally polymorphic in plants.

Retrotransposons and retroviruses share similar intracellular life cycles and major encoded proteins, but retrotransposons lack the envelope (env) critical for infectivity. Retrotransposons are ubiquitous and abundant in plants and active retroviruses are known in animals. Although a few env-containing retroelements, gypsy-like Athila, Cyclops, and Calypso and copia-like SIRE-1, have been identified in plants, the general presence and functionality of the domain remains unclear. We show here that env-class elements are present throughout the flowering plants and are widely transcribed. Within the grasses, we show the transcription of the env domain itself for Bagy-2 and related retrotransposons, all members of the Athila group. Furthermore, Bagy-2 transcripts undergo splicing to generate a subgenomic env product as do those of retroviruses. Transcription and the polymorphism of their insertion sites in closely related barley cultivars suggests that at least some are propagationally active. The putative ENV polypeptides of Bagy-2 and rice Rigy-2 contain predicted leucine zipper and transmembrane domains typical of retroviral ENVs. These findings raise the prospect of active retroviral agents among the plants.

Changes in gene expression in the leafy cotyledon1 (lec1) and fusca3 (fus3) mutants of Arabidopsis thaliana L.

Arabidopsis thaliana L. leafy cotyledon1 (lec1) and fusca3 (fus3) mutants show multiple phenotypic defects during seed development. In this report the effects of these mutations are examined at the molecular level. The patterns of protein accumulation in lec1 and fus3 seeds are severly altered. In lec1 seeds the steady-state mRNA levels of several late embryogenesis genes were reduced. Different patterns of expression were observed, indicating the occurrence of several regulatory pathways. The effect of lec1 mutations on the expression of the late-embryogenesis abundant AtEm1 gene was examined in detail. In lec1-1 seeds, the AtEm1 gene was expressed at a higher level than in the wild type and earlier in development. The activity of an AtEm1 promoter/beta-glucuronidase reporter gene construct in transgenic A. thaliana plants was studied. Changes in promoter activity in lec1-1 with respect to wild-type seeds were correlated with changes in corresponding mRNA steady-state levels. fus3-2 mutation produced similar changes in AtEm1 promoter activity as lec1-1, which is consistent with the hypothesis that LEC1 and FUS3 might act in the same regulatory pathway. Transgenic analysis using 5'-promoter deletions demonstrated that at least two regions of AtEm1 gene promoter interact with the LEC1-dependent transcriptional regulatory pathway. In spite of expression of the AtEm1 promoter and accumulation of AtEm1 mRNA, the corresponding Em1 protein does not accumulate in lec1-1 seeds. The ABA inducibility of the AtEm1 promoter was not affected by the lec1 mutation.

The Arabidopsis AtEm1 promoter is active in Brassica napus L. and is temporally and spatially regulated.

The promoter of the Arabidopsis thaliana L. AtEm1 gene encoding a late embryogenesis abundant protein was fused to the beta-glucuronidase reporter gene and introduced into Brassica napus. The promoter is highly active in the vascular tissues of embryo and pollen grains and also active in petals, sepals, caulinar leaves, and carpels.

What makes Grande1 retrotransposon different?

Grande1 elements constitute a family of Ty3 retrotransposons present in the Zea genus in more than 1000 copies in Zea diploperennis and maize. The sequences of three Grande1 flanking regions, two from Z. diploperennis and one from maize, reveal transposable elements as insertion targets, suggesting a preferential integration of Grande1 elements into other transposable elements. These retrotransposons are remarkable for their large size of around 14 kb, which is a consequence of a very large 3' region of more than 7 kb. Atypical entities within this region are two arrays of unrelated tandem repeats with potential stable stem-loop structures. A large portion of the same region is occupied by ORFs, although only ORF23, whose function is unknown, is presumably transcribed in antisense orientation to the reverse transcriptase ORF. Only ORF23 has a codon usage similar to the one tabulated for highly-expressed maize genes. Correspondingly, the transcript of 900 b that hybridizes with ORF23 probes is found in all the maize tissues explored. This is despite the high level of methylation in the DNA of Grande1. Genomic RNA has not been detected in any tissue or situation studied, probably reflecting a non-functional retrotransposon. The origin of ORF23 and the remainder 3' region might be due to a transduction event.

Active retrotransposons are a common feature of grass genomes.

A large fraction of the genomes of grasses, members of the family Graminae, is composed of retrotransposons. These elements resemble animal retroviruses in their structure and possess a life cycle similar to theirs that includes transcription, translation, and integration of daughter copies. We have investigated if retrotransposons are generally transcribed in the grasses and other plants, and whether the various families of elements are translationally and integrationally active in multiple grass species. A systematic search of 7.8 x 10(5) publicly available expressed sequence tags from plants revealed widespread retrotransposon transcripts at a frequency of one in 1,000. Monocot retrotransposons found relatively more expressed sequence tags from non-source species than did those of dicots. Antibodies were raised to the capsid protein, GAG, of BARE-1, a transcribed and translated copia-like retrotransposon of barley (Hordeum vulgare). These detected immunoreactive proteins of sizes identical to those of the BARE-1 GAG and polyprotein, respectively, in other species of the tribe Triticeae as well as in oats (Avena sativa) and rice (Oryza sativa). Retrotransposon-based markers showed integrational polymorphisms for BARE-1 in different subfamilies of the Graminae. The results suggest that grasses share families of transcriptionally, translationally, and integrationally active retrotransposons, enabling a comparative and integrative approach to understanding the life cycle of retrotransposons and their impact on the genome.

Differential expression of the Arabidopsis genes coding for Em-like proteins.

Late embryogenesis abundant (lea) genes are a large and diverse group of genes highly expressed during late stages of seed development. Five major groups of LEA proteins have been described. Two Em genes (group I lea genes) are present in the genome of Arabidopsis thaliana L., AtEm1 and AtEm6. Both genes encode for very similar proteins which differ basically in the number of repetitions of a highly hydrophilic amino acid motif. The spatial patterns of expression of the two Arabidopsis Em genes have been studied using in situ hybridization and transgenic plants transformed with the promoters of the genes fused to the beta-glucuronidase reporter gene (uidA). In the embryo, AtEm1 is preferentially expressed in the pro-vascular tissues and in meristems. In contrast, AtEm6 is expressed throughout the embryo. The activity of both promoters disappears rapidly after germination, but is ABA-inducible in roots of young seedlings, although in different cells: the AtEm1 promoter is active in the internal tissues (vasculature and pericycle) whereas the AtEm6 promoter is active in the external tissues (cortex, epidermis and root hairs). The AtEm1 promoter, but not AtEm6, is also active in mature pollen grains and collapsed nectaries of young siliques. These data indicate that the two Em proteins could carry out at least slightly different functions and that the expression of AtEm1 and AtEm6 is controlled at, at least, three different levels: temporal, spatial and hormonal (ABA).

Structure, functionality, and evolution of the BARE-1 retrotransposon of barley.

The BARE-1 retrotransposon is a major, active component of the genome of barley (Hordeum vulgare L.) and other Hordeum species. Copia-like in its organization, it consists of 1.8-kb long terminal repeats bounding an internal domain of 5275 bp which encodes a predicted polyprotein of 1301 residues. The polyprotein contains the key residues, structural motifs, and conserved regions associated with retroviral and retrotransposon GAG, aspartic proteinase, integrase, reverse transcriptase, and RNaseH polypeptides. BARE-1 is actively transcribed and translated. As part of our effort to understand the evolution and function of BARE-1, we have examined its copy number and localization. Full-length members of the BARE-1 family constitute 2.8% of the barley genome. Globally, they are dispersed throughout the genome, excepting the centromeric, telomeric, and NOR regions. Locally, BARE-1 occurs more commonly in repetitive DNA than in coding regions, forming clusters of nested insertions. Both barley and other Hordeum genomes contain a high proportion of BARE-1 solo LTRs. New techniques have been developed which exploit the insertion site polymorphism generated by BARE-1 integration to produce molecular markers for breeding, biodiversity, and mapping applications.

Chromosome localization and characterization of a family of long interspersed repetitive DNA elements from the genus Zea.

This paper describes the characterization and chromosomal distribution of new long repetitive sequences present in all species of the genus Zea. These sequences constitute a family of moderately repetitive elements ranging approximately from 1350 to 1700 copies per haploid genome in modern maize (Zea mays ssp. mays) and teosinte (Zea diploperennis), respectively. The elements are long, probably larger than 9 kb, and they show a highly conserved internal organization among Zea subspecies and species. The elements are present in all maize chromosomes in an interspersed pattern of distribution, are absent from centromeric and pericentric heterochromatin, and with some clustering in the distal regions of chromosome arms.