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53 pages, 10 figures, 20 tables, This paper provides an overview of the last 40 years of use, and in many cases abuse, of the natural resources in Catalonia, a country that is representative of European countries in general, and especially those in the Mediterranean region. It analyses the use of natural resources made by mining, agriculture, livestock, logging, fishing, nature tourism, and energy production and consumption. This use results in an ecological footprint, i.e., the productive land and sea surface required to generate the consumed resources and absorb the resulting waste, which is about seven times the amount available, a very high number but very similar to other European countries. This overexploitation of natural resources has a huge impact on land and its different forms of cover, air, and water. For the last 25 years, forests and urban areas have each gained almost 3% more of the territory at the expense of agricultural land; those municipalities bordering the sea have increased their number of inhabitants and activity, and although they only occupy 6.7% of the total surface area, they account for 43.3% of the population; air quality has stabilized since the turn of the century, and there has been some improvement in the state of aquatic ecosystems, but still only 36% are in good condition, while the remainder have suffered morphological changes and different forms of nonpoint source pollution; meanwhile the biodiversity of flora and fauna remains still under threat. Environmental policies do not go far enough so there is a need for revision of the legislation related to environmental impact and the protection of natural areas, flora, and fauna. The promotion of environmental research must be accompanied by environmental education to foster a society which is more knowledgeable, has more control and influence over the decisions that deeply affect it. Indeed, nature conservation goes hand in hand with other social and economic challenges that require a more sustainable vision. Today’s problems with nature derive from the current economic model, which is environmentally unsustainable in that it does not take into account environmental impacts. Lastly, we propose a series of reasonable and feasible priority measures and actions related to each use made of the country’s natural resources, to the impacts they have had, and to their management, in the hope that these can contribute to improving the conservation and management of the environment and biodiversity and move towards sustainability, This research was funded by the Institut d’Estudis Catalans (IEC). With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

13 páginas, 4 figuras, 3 tablas., [Background]: Cucumis melo (melon) belongs to the Cucurbitaceae family, whose economic importance among horticulture crops is second only to Solanaceae. Melon has high intra-specific genetic variation, morphologic diversity and a small genome size (450 Mb), which make this species suitable for a great variety of molecular and genetic studies that can lead to the development of tools for breeding varieties of the species. A number of genetic and genomic resources have already been developed, such as several genetic maps and BAC genomic libraries. These tools are essential for the construction of a physical map, a valuable resource for map-based cloning, comparative genomics and assembly of whole genome sequencing data. However, no physical map of any Cucurbitaceae has yet been developed. A project has recently been started to sequence the complete melon genome following a whole-genome shotgun strategy, which makes use of massive sequencing data. A BAC-based melon physical map will be a useful tool to help assemble and refine the draft genome data that is being produced. [Results]: A melon physical map was constructed using a 5.7 × BAC library and a genetic map previously developed in our laboratories. High-information-content fingerprinting (HICF) was carried out on 23,040 BAC clones, digesting with five restriction enzymes and SNaPshot labeling, followed by contig assembly with FPC software. The physical map has 1,355 contigs and 441 singletons, with an estimated physical length of 407 Mb (0.9 × coverage of the genome) and the longest contig being 3.2 Mb. The anchoring of 845 BAC clones to 178 genetic markers (100 RFLPs, 76 SNPs and 2 SSRs) also allowed the genetic positioning of 183 physical map contigs/singletons, representing 55 Mb (12%) of the melon genome, to individual chromosomal loci. The melon FPC database is available for download at http://melonomics.upv.es/static/files/public/physical_map/ webcite. [Conclusions]: Here we report the construction of the first physical map of a Cucurbitaceae species described so far. The physical map was integrated with the genetic map so that a number of physical contigs, representing 12% of the melon genome, could be anchored to known genetic positions. The data presented is already helping to improve the quality of the melon genomic sequence available as a result of a project currently being carried out in Spain, adopting a whole genome shotgun approach based on 454 sequencing data., This project was funded by the Plan Nacional de Investigación Científica of the Spanish Ministerio de Educación y Ciencia (Project BIO2007-61789) and by the Consolider-Ingenio 2010 Programme of the Spanish Ministerio de Ciencia e Innovación (CSD2007-00036 "Center for Research in Agrigenomics").

Bioeconomy, biotechnology and genetically-modified organisms in particular have been the subject of discussion for a long time. Biotechnology is applied in a variety of economic areas which include biopharmaceuticals, biobased products and agriculture. During the last 20 years, innovative biotechnological techniques for plant genome improvement have been developed. Many factors worldwide have led to the status quo : different legislations around the world, the lack of public acceptance in the EU and high expectations for new strategies for sustainability and food security. Therefore, a clear regulatory status for new techniques is crucial for research and development, as well as for their practical implementation. This should be based on solid science which plays a critical role in developing the bioeconomy., This paper was partially supported by both the Polish Ministry of Science and Higher Education within the KNOW program and a grant for young scholars as well as the Polish Ministry of Agriculture and Rural Development within the multiannual program 2015-2020.

Genetically modified (GM) maize and their non-modified counterparts were compared using MON810 varieties, the only GMO event cultivated in Europe. The differences in grain samples were analysed by omics profiles, including transcriptomics, proteomics and metabolomics. Other cultivated maize varieties were analysed as a reference for the variability that will exist between cultivated varieties. The observed differences between modified and non-modified maize varieties do not exceed typical differences between non-modified varieties. The use of these advanced analytical approaches to analyse novel plant materials as compared to the results from animal feeding trials with whole foods is assessed. No indications were observed for changes in the GM varieties that warrant further investigations. Furthermore, it was shown that such indications will be obtained if maize samples of inferior quality are analysed similarly. Omics data provide detailed analytical information of the plant material, which facilitates a risk assessment procedure of new (GM) plant varieties., This study was carried out as part of the GRACE project (“GMO Risk Assessment and Communication of Evidence”), financially supported by the 7th Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (FP7), Grant Agreement No. 311957.

The insect resistant maize YieldGard MON810 was studied to assess the extent to which introduction of a transgene may putatively alter the expression of endogenous genes by comparison of various GM lines vs. their non-transgenic counterparts. To assess the extent to which introduction of a transgene may putatively alter the expression of endogenous genes, GM lines of the insect resistant maize YieldGard MON810 were compared with non-transgenic counterparts. For a more in-depth study, high-throughput deep sequencing together with microarrays were used to compare the transcriptomes of immature embryos of the MON810 variety DKC6575, with a cryIA(b) transgene, and its near-isogenic variety Tietar, grown under controlled environmental conditions. This technique also allows characterisation of the transgenic mRNAs produced. 3′UTR-anchored mRNA-seq produced 1,802,571 sequences from DKC6575 and 1,170,973 from Tietar, which mapped to 14,712 and 14,854 unigenes, respectively. Up to 32 reads from the transgenic embryos matched to the synthetic cry1A(b) sequence, similar to medium-abundant mRNAs. Gene expression analysis, using the R-bioconductor packages EdgeR and DEseq, revealed 140 differentially expressed genes mainly involved in carbohydrate metabolism, protein metabolism and chromatin organisation. Comparison of the expression of 30 selected genes in two additional MON810 and near-isogenic variety pairs showed that most of them were differentially expressed in the three pairs of varieties analysed. Analysis of functional annotation and the precise moment of expression of the differentially expressed genes and physiological data obtained suggest a slight but significant delay in seed and plant maturation of MON810 plants. However, these transcriptomic changes were not associated to undesirable changes in the global phenotype or plant behaviour. Moreover, while most expression changes in MON810 immature embryos were maintained in other transgenic varieties, some gene expression was found to be modulated by the genetic background in which the transgene was introduced through conventional breeding programs., This work was funded by the Spanish MEC project ref. AGL2007-65903/AGR, the CONSOLIDER project and the Xarxa de Referència en Biotecnología of the Generalitat de Catalunya.

Podemos considerar la Biotecnología Vegetal tan antigua como la misma acción de los humanos sobre los vegetales con el objetivo de obtener de ellos productos para la alimentación u otros usos, por tanto tan antigua como la Agricultura. Sin embargo ha sido en el siglo XXcon la introducción de la Genética primero y de la Biología Molecular después, que se ha desarrollado esta disciplina en su forma actual. Desde 1983 ha sido posible obtener plantas con modificaciones genéticas obtenidas con métodos moleculares. Estas plantas han sido el objeto de regulaciones especiales y de un intenso debate. El desarrollo de la Genómica permite la extensión de estas aplicaciones y de otras que no requieren de técnicas de transformación.Encualquier caso, la Biotecnología Vegetal ha demostrado su utilidad para responder a los retos que la producción agrícola tiene en el próximo futuro. La producción de alimentos y otros materiales en un entorno de crecimiento de la población y de cambios en el clima está necesitando utilizar todo el conocimiento disponible en la actualidad.

We sequenced the genome of the highly heterozygous almond Prunus dulcis cv. Texas combining short and long-read sequencing. We obtained a genome assembly totaling 227.6 Mb of the estimated 238 Mb almond genome size, of which 91% is anchored to eight pseudomolecules corresponding to its haploid chromosome complement, and annotated 27,969 protein-coding genes and 6,747 non-coding transcripts. By phylogenomic comparison with the genomes of 16 additional close and distant species we estimated that almond and peach (P. persica) diverged around 5.88 Mya. These two genomes are highly syntenic and show a high degree of sequence conservation (20 nucleotide substitutions/kb). However, they also exhibit a high number of presence/absence variants, many attributable to the movement of transposable elements (TEs). TEs have generated an important number of presence/absence variants between almond and peach, and we show that the recent history of TE movement seems markedly different between them. TEs may also be at the origin of important phenotypic differences between both species, and in particular, for the sweet kernel phenotype, a key agronomic and domestication character for almond. Here we show that in sweet almond cultivars, highly methylated TE insertions surround a gene involved in the biosynthesis of amygdalin, whose reduced expression has been correlated with the sweet almond phenotype. Altogether, our results suggest a key role of TEs in the recent history and diversification of almond and its close relative peach., This research was supported in part by grants from: the Ministry of Economy and Competitiveness (MINECO/FEDER projects AGL2012-40228-C02-01, AGL2015-68329-R and RTA2015-00050-00-00, Severo Ochoa Program for Centres of Excellence in R&D 201-2019 SEV-2015-0533 and CERCA Programme-Generalitat de Catalunya) from Spain. MW acknowledges grant support from the Waite Research Institute of the University of Adelaide.

Combining both short and long-read sequencing, we have estimated the almondPrunus dulciscv. Texas genome size in 235 Mbp and assembled 227.6 Mb of its sequence. The highly heterozygous compact genome of Texas comprises eight chromosomes, to which we have anchored over 91% of the assembly. We annotated 27,042 protein-coding genes and 6,800 non-coding transcripts. High levels of genetic variability were characterized after resequencing a collection of ten almond accessions. Phylogenomic comparison with the genomes of 16 other close and distant species allowed estimating that almond and peach diverged around 5.88 Mya. Comparison between peach and almond genomes confirmed the high synteny between these close relatives, but also revealed high numbers of presence-absence variants, many attributable to the movement of transposable elements (TEs). The number and distribution of TEs between peach and almond was similar, but the history of TE movement was distinct, with peach having a larger proportion of recent transpositions and almond preserving a higher level of polymorphism in the older TEs. When focusing on specific genes involved in key characters such as the bitter vs. sweet kernel taste and the formation of a fleshy mesocarp, we found that for one gene associated with the biosynthesis of amygdalin that confers the bitter kernel taste, several TEs were inserted in its vicinity only in sweet almond cultivars but not in bitter cultivars andPrunusbitter kernel relatives, includingP. webbii,P. mume, and other species like peach and cherry. TE insertions likely to produce affects in the expression of six more genes involved in the formation of the fleshy mesocarp were also identified. Altogether, our results suggest a key role of TEs in the recent history and diversification of almond with respect to peach.

The authors have been members of the EFSA GMO Panel (P.P. 2003–2006, J.C. 2006–2012 and 2015–2018). Pere Puigdomènech has been member of the GRACE (2012–2015) and G-TwYST (2014–2018) projects funded by the EU FP7 Program.

We report an improved assembly (v3.6.1) of the melon (Cucumis melo L.) genome and a new genome annotation (v4.0). The optical mapping approach allowed correcting the order and the orientation of 21 previous scaffolds and permitted to correctly define the gap-size extension along the 12 pseudomolecules. A new comprehensive annotation was also built in order to update the previous annotation v3.5.1, released more than six years ago. Using an integrative annotation pipeline, based on exhaustive RNA-Seq collections and ad-hoc transposable element annotation, we identified 29,980 protein-coding loci. Compared to the previous version, the v4.0 annotation improved gene models in terms of completeness of gene structure, UTR regions definition, intron-exon junctions and reduction of fragmented genes. More than 8,000 new genes were identified, one third of them being well supported by RNA-Seq data. To make all the new resources easily exploitable and completely available for the scientific community, a redesigned Melonomics genomic platform was released at http://melonomics.net. The resources produced in this work considerably increase the reliability of the melon genome assembly and resolution of the gene models paving the way for further studies in melon and related species., This work was supported by the Spanish Ministry of Economy and Competitiveness grant AGL2015–64625-C2-1-R, Severo Ochoa Programme for Centres of Excellence in R&D 2016–2010 (SEV-2015–0533) and the CERCA Programme/Generalitat de Catalunya to JGM. VR was supported by the European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement No 665919.

Few regulators of phenylpropanoids have been identified in monocots having potential as biofuel crops. Here we demonstrate the role of the maize (Zea mays) R2R3-MYB factor ZmMYB31 in the control of the phenylpropanoid pathway. We determined its in vitro consensus DNA-binding sequence as ACC(T)/(A) ACC, and chromatin immunoprecipitation (ChIP) established that it interacts with two lignin gene promoters in vivo. To explore the potential of ZmMYB31 as a regulator of phenylpropanoids in other plants, its role in the regulation of the phenylpropanoid pathway was further investigated in Arabidopsis thaliana. ZmMYB31 downregulates several genes involved in the synthesis of monolignols and transgenic plants are dwarf and show a significantly reduced lignin content with unaltered polymer composition. We demonstrate that these changes increase cell wall degradability of the transgenic plants. In addition, ZmMYB31 represses the synthesis of sinapoylmalate, resulting in plants that are more sensitive to UV irradiation, and induces several stress-related proteins. Our results suggest that, as an indirect effect of repression of lignin biosynthesis, transgenic plants redirect carbon flux towards the biosynthesis of anthocyanins. Thus, ZmMYB31 can be considered a good candidate for the manipulation of lignin biosynthesis in biotechnological applications.

The involvement of the maize ZmMYB42 R2R3-MYB factor in the phenylpropanoid pathway and cell wall structure and composition was investigated by overexpression in Arabidopsis thaliana. ZmMYB42 down-regulates several genes of the lignin pathway and this effect reduces the lignin content in all lignified tissues. In addition, ZmMYB42 plants generate a lignin polymer with a decreased S to G ratio through the enrichment in H and G subunits and depletion in S subunits. This transcription factor also regulates other genes involved in the synthesis of sinapate esters and flavonoids. Furthermore, ZmMYB42 affects the cell wall structure and degradability, and its polysaccharide composition. Together, these results suggest that ZmMYB42 may be part of the regulatory network controlling the phenylpropanoid biosynthetic pathway., This work received financial support from the CONSOLIDER-INGENIO programme (CSD2007-00036) from the Spanish Ministerio de Ciencia e Innovación. F.M. Sonbol was financed by the European Project INCO-II (ICA4-CT-2000-30017), by the CRAG laboratory, and by the “Instituto de Cooperación con el Mundo Árabe, Agencia Española de Cooperación Internacional”. S. Fornale was financed by a post-doctoral grant from the “Generalitat de Catalunya” (2003PIV-A-00033) and by an I3P contract from the “Consejo Superior de Investigaciones Científicas”. D. Caparrós-Ruiz was financed by the Spanish “Ministerio de Educación y Ciencia” (“Ramón y Cajal” Program). This work was carried out within the framework of the “Xarxa de Referència de Biotecnologia” (XarBa) from the Autonomous Government of Catalonia.

In the past years, there has been a great effort to obtain genomic tools in melon (Cucumis melo L.). Advances in next-generation sequencing technologies have made possible to obtain de novo draft genome sequences for many plant species, including melon and other cucurbits. Since the publication of the cucumber, melon and watermelon genomes, the cucurbit scientific community has a collection of powerful tools that can be used to help understand important biological processes that are unique to these species. At the same time, genome sequences can be used to assist in breeding programs for the development of improved varieties. This chapter describes the collection of genetic and genomic tools that have been developed in melon until the publication of the melon genome, some of which have been centralized at the ICuGI webpage. The main features of the published melon genome, some data about comparative genomic studies among cucurbit species and some possible applications of the melon genome sequence are also discussed.

The availability of next-generation sequencing technologies in the past years has allowed unprecedented access to draft genome sequences for the main crops and plants. These genome sequences offer new possibilities for studying genome evolution, for understanding the biological processes controlling important traits and for improving plant breeding. Among them, the genome of melon (Cucumis melo L.), one of the main cucurbit crops, was released in 2012. Since the publication of the reference genome of the double haploid line DHL92 v3.5, the genome assembly and annotation have been improved in new genome versions. The melon genome has been useful for performing several comparative analysis with other cucurbit genomes, for analysing the genome structure and extant variability and for the isolation and characterization of several important genes.

A real-time polymerase chain reaction (QPCR) assay was developed for quantitative detection of a genetically modified (GM) maize event TC-1507 and modification to conventional PCR for qualitative purposes. Sequences 5-flanking TC-1507 full-length insert were characterized and showed multiple rearrangements involving insert and maize chloroplast fragments. The event specificity of the TC-1507 assays was based on the detection of transgene and plant rearranged sequences found to 5 flank the insertion site. They were fully specific and exhibited a limit of detection below 10 target copies, allowing consistent detection of 0.1% GM levels. The QPCR was highly linear and efficient and proved adequate for quantification of GM contents, aiming at the fulfillment of legal requirements established in the European Union (i.e., compulsory labeling of TC-1507 levels >0.9%). It satisfactorily determined TC-1507 contents on different matrixes and was successfully transferred to a different laboratory.

Plant laccases are enzymes that have been proposed to participate in the last step of lignin biosynthesis. The polymerisation event remains still much unknown, implicating other enzymes such as peroxidases. To gain more insight in how this polymerisation process takes place in maize, we isolated by differential screening of an elongation maize root cDNA library four cDNA clones encoding a family of laccases. Three of them (ZmLac2, ZmLac4, ZmLac5) were basic enzymes, while one of them (ZmLac3) was an acidic enzyme. Southern analysis indicates that laccases belong to a multigene family in maize. Phylogenetic analysis reveals that ZmLac2, ZmLac4, and ZmLac5 are closely related enzymes, whereas ZmLac3 is a slightly different enzyme. The pattern of mRNA accumulation of ZmLac2, ZmLac3, ZmLac4, and ZmLac5 genes correlates with the maize regions undergoing lignification. Moreover, ZmLac3 is induced by wounding, whereas ZmLac2 and ZmLac5 are repressed and ZmLac4 gene expression is not affected. Taken together, our results suggest that the acidic ZmLac3 enzyme could be involved in the polymerisation of phenolic compounds in maize. Instead, and in agreement with the idea that laccases are enzymes involved in a wide range of physiological processes, results obtained with ZmLac2, ZmLac4, and ZmLac5 lead us to exclude a direct role of these laccases in lignin polymerisation., This work has been funded by the Spanish “Ministerio de Ciencia y Tecnología” (BIO2001-1140). We are indebted to the sequencing team of IBMB-CSIC as well as Dr. Castresana (IBMB-CSIC) for his advices and comments on phylogenetic analyses performed in this work. D-C.R was initially financed by the European COPOL Project QLRT-1999-31493 and later on by the Spanish “Ministerio de Educacion y Ciencia” (“Ramon y Cajal” Program). S-F was financed by a post-doctoral grant of the “Generalitat de Catalunya” (2004-CRED-10005). We are indebted to Dr. Burgess for the English correction of this manuscript. This work was carried out within the framework of the Centre de Referència de Biotecnologia (CeRBA) from the Generalitat de Catalunya.

Plant genomes contain numerous genes (R-genes) that play a role in initiating defence measures against their particular pathogens. Defence mechanisms controlled by R-genes have been the focus of extensive research over the past several years. The majority of the R-genes described so far belong to a super-family of genes (150-600 members) that encode proteins with a nucleotide binding site (NBS), some leucine-rich repeats (LRR) and an N-terminal domain that shows similarity to the Toll and Interleukin-1 receptors (TIR) or a N-terminal coiled-coil (CC) domain. Analysis of four regions of the melon (Cucumis melo) genome, including two sequenced BACs, identified 14 TIR-NBS-LRR genes. Known disease resistance genes have been mapped in three of these regions. Transcriptional expression was detected for predicted genes that are possibly involved in defence responses to pathogen attack. TIR-NBS-LRR genes appear to be clustered in the melon genome. They contain all the conserved motifs that have previously been described for their counterparts in other species, although differences were also detected. The results presented here may contribute to a better understanding of the genomic distribution and evolution of this group of resistance gene homologues and their variability. © Springer-Verlag 2005., Funds for this project were provided by Grant No. 2FD97-0286-(02–01) from the Comisión Interdepartamental de Ciencia y Tecnologia (CICYT) and Grant No. 2000FI-00316 to Hans van Leeuwen from the Comissió In- terdepartamental de Recerca i Tecnologia (CIRIT). This work was carried out within the context of the Centre de Referencia de Biotecnologia (CERBA) of the Generalitat de Catalunya.

In this paper, a method for the specific detection and quantification of potato and tomato DNA in food samples with the use of conventional and real-time polymerase chain reaction (PCR) is described. This method is adequate for use in food quality routine assays involving highly processed samples for which very tiny amounts of DNA are expected. Detection was achieved by amplifying a region of the metallo-carboxypeptidase inhibitor gene from either the potato (PCI) or the tomato (MCPI) and by using specific primers complementary to the propeptide regions of these inhibitors, which were found to differ for the potato and tomato proproteins. Conventional and real-time PCR systems were based on the same potato- or tomato-specific primer pairs, and quantification was carried out with a TaqMan chemistry-based probe. The methods developed proved to be very specific and sensitive and highly reliable for the identification and quantification of DNA from both plant species. In addition, the construction of plasmids pPAT and pTOM, suitable for use as external calibration standards for the elaboration of comparative amplification profiles, is reported. Limits of detection and quantification with the use of these plasmid standards are given. Specificity and copy number conservation among different cultivars were analyzed, and the reliability of these systems was tested through their application to the analysis of commercial food samples including potato and/or tomato as components.

The transcript population of the human gene coding for a cGMP-dependent phosphodiesterase (PDE9A) has a complex structure. There is a high level of mRNA in intestinal and prostate tissues, a low level in blood, and intermediate in other tissues. More than 20 different variants produced by differential splicing have been observed and new exons have been identified both by PCR amplification and by the analysis of available EST sequences. In all cases the transcriptional start site is the same and no differential splicing is found in the exons coding for the catalytic domain of the protein. In some cases the protein produced by splice variants is truncated. The distribution of the splice variants is not homogeneous among the different tissues studied. The human, but not the mouse, PDE9A gene appears to have a complex regulation of expression by different isoforms.

A bacterial artificial chromosome (BAC) library from the dihaploid melon line 'PIT92' was constructed with a 6 times coverage of the haploid melon genome. A contig of four BACs around the MRGH63 resistance gene homologue fragment was created. The complete sequence of a 117-kb BAC clone allowed to determine two clearly defined regions, the first one containing a cluster of three resistance gene homologues. Separated by a retrotransposon, that contains large long terminal repeats, the second region presents a group of genes with a conserved distribution in two regions of the Arabidopsis genome. The detailed analysis of this region provides a description of the gene structure and the presence of repetitive sequences in a defined fragment of the genome of Cucumis melo.

The increasing presence of transgenic plant derivatives in a wide range of animal and human consumables has provoked in western Europe a strong demand for appropriate detection methods to evaluate the existence of transgenic elements. Among the different techniques currently used, the real-time quantitative PCR is a powerful technology well adapted to the mandatory labeling requirements in the European Union (EU). The use of transgene flanking genomic sequences has recently been suggested as a means to avoid ambiguous results both in qualitative and quantitative PCR-based technologies. In this study we report the identification of genomic sequences adjacent to the 3'-integration site of event MON810 in transgenic maize. This genetically modified crop contains transgene sequences leading to ectopic expression of a synthetic CryIA(b) endotoxin which confers resistance to lepidopteran insects especially against the European corn borer. The characterization of the genome-transgene junction sequences by means of TAIL-PCR has facilitated the design of a specific, sensitive and accurate quantification method based on TaqMan chemistry. Cloning of event MON810 3'-junction region has also allowed to compare the suitability of plasmid target sequences versus genomic DNA obtained from certified reference materials (CRMs), to prepare standard calibration curves for quantification.

An α-L-fucosidase purified from pea (Pisum sativum L. cv Alaska) epicotyl was previously described as a cell wall enzyme of 20 kDa that hydrolyses terminal α-L-fucosidic linkages from oligosaccharide fragments of xyloglucan. cDNA and genomic copies were further isolated and sequenced. The predicted product of the cDNA and the genomic clone (fuc1), was a 20 kDa protein containing a signal peptide and five cysteines. This was the first α-L-fucosidase gene to be cloned in plants but its fucosidase activity has not been demonstrated. Here, our biochemical and immuno analyses suggest that fuc1 does not encode an α-L-fucosidase. Pea fuc1 expressed in Escherichia coli, insect cells and Arabidopsis thaliana produced recombinant proteins without α-L-fucosidase activity. Pea plants had endogenous α-L-fucosidase activity, but the enzyme was not recognised by an antibody produced against recombinant FUC1 protein expressed in E. coli. In contrast, the antibody immunoprecipitated a 20 kDa protein which was inactive. By chromatographic analysis of pea protein extracts, we separated α-L-fucosidase-active fractions from the 20 kDa protein fractions. We conclude that the α-L-fucosidase activity is not attributable to the 20 kDa FUC1 protein. A new function for fuc1 gene product, now named PIP20 (for protease inhibitor from pea) is proposed.

cAMP plays an important role as a second-messenger molecule controlling multiple cellular processes. Its hydrolysis provides an important mechanism by which cAMP levels are regulated. This is performed by a large multigene family of cyclic nucleotide phosphodiesterases (PDEs). Members of the PDE4 enzyme family are selectively inhibited by rolipram. Five different mRNA splice forms for PDE4D have been isolated. Here, we analyzed the regional distribution of the mRNAs coding for the splice variants PDE4D1, PDE4D2, PDE4D3, PDE4D4, and PDE4D5 in the rat brain by in situ hybridization histochemistry using specific radiolabeled oligonucleotides. We found that all five splice variants showed a distinct distribution pattern and, in some cases, in association with specific brain pathways. The most relevant differences were in hippocampal formation, medial habenula, basal ganglia, and area postrema, at both the regional and cellular level. The dorsal and median raphe nuclei exclusively contained PDE4D2 mRNA transcripts, probably located on serotonergic cells. PDE4D1 mRNA was expressed in some white matter cells. PDE4D1 and PDE4D2 mRNA splice forms presented a similar distribution in the area postrema, whereas for PDE4D4 and PDE4D5 the cellular distribution presented a complementary pattern. The differential expression of PDE4D mRNA splice variants in the area postrema is consistent with their possible involvement in emesis control and suggests new molecular targets for a more selective drug design.

The maize cDNA sequence (ZmPRL) homologous to the Arabidopsis thaliana PROLIFERA or to the different MCM7 genes (yeast cdc47, as an example) has been identified by differential display due to its increased mRNA accumulation in the apical region of the maize immature embryo. Probably two sequences coding for ZmPRL exist in the maize genome and when comparing the different MCM protein sequences available, the protein clusters with other MCM7 proteins already described. Analysis by in situ hybridization shows that the pattern of ZmPRL mRNA accumulation is in agreement with the pattern observed for genes related to cell cycle in plants and that the expression is mostly found in the apical meristem. It is proposed that this is a consequence of the mechanisms that control the formation of the apical-basal pattern in plant embryo acting upon the regulation of the cell cycle.

Four families of resistance gene homologues containing the nucleotide binding site (NBS) motif were obtained in Cucumis melo using degenerate primers designed from two conserved regions of known disease resistance genes. The four homologues had high levels of amino acid identity to NBS-containing resistance genes found in other species. Three of these homologues (MRGH4, MRGH21 and MRGH63) were placed in the genetic map of melon. The RFLP detected with MRGH21 was located on the distal part of Linkage Group 7. Homologues MRGH63 and MRGH4 detected RFLPs that mapped to Linkage Group 4. The map positions of these loci relative to those of resistance genes mapped in melon and the potential use of these loci in marker-assisted selection and map-based cloning strategies is discussed., Funds for this project were provided by grant 2FD97-0286-C02-01 from the Spanish ‘Comisión Interdepartamental de Ciencia y Tecnologı́a’ (CICYT).

This work was supported by grants from Fundacio La Marato de TV3 (1017/97), and CICYT (SAF1999-0123 and 2FD97-0395). X.M. is a recipient of a fellowship from CIRIT (Centre de Referencia of the Generalitat de Catalunya) and S.P.-T. from CIRIT (Generalitat de Catalunya)

We have examined the distribution of four different cyclic AMP-specific phosphodiesterase isozyme (PDE4A, PDE4B, PDE4C and PDE4D) mRNAs in the brain of different species by in situ hybridization histochemistry and by autoradiography with [3H]rolipram. We have compared the localization of each isozyme in human brain with that in rat and monkey brain. We have found that the four PDE4 isoforms display a differential expression pattern at both regional and cellular level in the three species. PDE4A, PDE4B and PDE4D are widely distributed in human brain, with the two latter appearing more abundant. In contrast, PDE4C in human brain, presents a more restricted distribution, limited to cortex, some thalamic nuclei and cerebellum. This is at variance with the distribution of PDE4C in rat brain, where it is found exclusively in olfactory bulb. In monkey brain, the highest expression for this isoform is found in the claustrum, and at lower levels in cortical areas and cerebellum. PDE4B presented a broad distribution, being expressed in both neuronal and non neuronal cell populations. In general, the distribution of binding sites visualized with [3H]rolipram correlated well with the expression of each PDE4 isozyme.

The distribution of repetitive sequences, or microsatellites, formed by either one or two base pairs and longer than eight units, has been studied in almost 1 Mb of the sequenced Arabidopsis thaliana genome. Except for those formed by only G and C residues, the repetitions are more abundant in the Arabidopsis genome than can be calculated from its nucleotide composition. They are distributed in proportions higher than expected in introns, and in the intergenic regions both proximal and distal to the coding sequences. In exons, only the TC/GA microsatellite seems to be particularly abundant. The AT/TA microsatellites produce more length variation between Arabidopsis ecotypes than the A/T repeated sequences. These two classes are more abundant per kilobase than coding sequences in the Arabidopsis genome. The results indicate not only that the presence of microsatellites is not an effect of random distribution of nucleotides, but that their resolution as molecular markers may be equivalent to the number of genes and also that they do not seem to be systematically linked to specific regulatory sequences proximal to genes.

At least two main groups of glycoproteins can be distinguished in plant cell walls: extensins which are insoluble cell wall proteins; and soluble arabinogalactan proteins (AGPs) which have a high carbohydrate content such that protein content constitutes in some cases only 5 % of the glycoprotein weight. These two groups of proteins together with other cell wall proteins more or less glycosylated, such as proline-rich proteins (PRPs), hybrid PRP (HyPRPs) and expansins, are reviewed and compared with similar proteins present in other cell compartments. Different patterns of N- or O-glycosylation are analysed. In some cases, these cell wall proteins or proteins related to them present patterns of glycosylation that act as epitopes recognizable by IgE in allergic responses.

Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25 000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.

The higher plant Arabidopsis thaliana (Arabidopsis) is an important model for identifying plant genes and determining their function. To assist biological investigations and to define chromosome structure, a coordinated effort to sequence the Arabidopsis genome was initiated in late 1996. Here we report one of the first milestones of this project, the sequence of chromosome 4. Analysis of 17.38 megabases of unique sequence, representing about 17% of the genome, reveals 3,744 protein coding genes, 81 transfer RNAs and numerous repeat elements. Heterochromatic regions surrounding the putative centromere, which has not yet been completely sequenced, are characterized by an increased frequency of a variety of repeats, new repeats, reduced recombination, lowered gene density and lowered gene expression. Roughly 60% of the predicted protein-coding genes have been functionally characterized on the basis of their homology to known genes. Many genes encode predicted proteins that are homologous to human and Caenorhabditis elegans proteins.

Hybridisation bands of mobility lower than expected appear in RNA blot analysis performed using total maize RNA hybridised with an hydroxyproline-rich glycoprotein (HRGP) cDNA probe. This behaviour has previously been observed and some authors have proposed that these bands could be transcripts of genes coding for highly similar genes. However, other data indicate that a single gene coding for HRGP exists in the maize genome. Here, we show that some of the mRNA molecules hybridising with the Hrgp probe migrate in agarose gel electrophoresis at a lower velocity than expected. The relative intensity of these low-mobility bands depends on the abundance of the Hrgp mRNA molecules loaded in the electrophoresis gels, a result confirmed by using two-dimensional gel electrophoresis. We propose that these hybridisation bands may be the product of different secondary structures or multimers of the 1.5-kb Hrgp mRNA probably due to high G+C content and the highly repetitive sequence of these molecules.

Full-length cDNAs encoding the monolignol biosynthetic enzymes caffeic acid 3-O-methyltransferase (COMT, EC 2.1.1.6), cinnamoyl-CoA reductase (CCR, EC 1.2.1.44) and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) were cloned from sugarcane (Saccharum officinarum L.). The encoded proteins (39.6, 40.1 and 38.7 kDa for COMT, CCR and CAD, respectively) were identified based on their sequence identities with the corresponding enzymes from other plant species. Pairwise comparisons of deduced amino acid sequences with known plant lignification proteins allowed the identification of important conserved domains and specific functional motifs within these enzymes. Two new conserved domains, probably involved in substrate specificity, are described for COMTs. Phylogenetic analysis showed a very close evolutionary relationship between sugarcane and maize sequences. Southern blot analyses are consistent with the presence of at least two copies of each studied gene in sugarcane genome. The comt, ccr and cad transcripts appear to happen in a parallel way in different sugarcane tissues. The mRNA accumulation patterns suggest a transcriptional regulation of these genes dependent on their specific role in lignin synthesis. The cloning and characterisation of sugarcane genes involved in lignification opens up the possibility of producing plants with lower and/or modified lignin by genetic engineering means.

A cDNA clone, PAB14B, was isolated from an almond flower cDNA library. The 1245 bp nucleotide sequence contains an open reading frame encoding a polypeptide of 356 amino acids which shows sequence similarity to O-methyltransferases (OMTs) from different plant species. The highest identity (49%) was observed with a methyltransferase from pea that catalyses the terminal step in pisatin phytoalexin biosynthesis. Comparison of this almond sequence, with amino acid sequences of described OMTs, identified regions of similarity which probably contribute to the binding site of S-adenosyl- l -methionine. Southern blot analysis suggests that PAB14B may be encoded by more than one gene in the almond genome. Flower-specific expression was shown by RNA blot analysis and the OMT transcript was detected in epidermal and vascular floral tissues by in situ hybridization. The available data suggest that this enzyme may be involved in the flavonoid biosynthesis pathway in almond floral tissues.

The pattern of expression of two genes coding for proteins rich in proline, HyPRP(hybrid proline-rich protein) and HRGP(hydroxyproline-rich glycoprotein), has been studied in maize (Zea mays) embryos by RNA analysis and in situ hybridization. mRNA accumulation is high during the first 20 d after pollination, and disappears in the maturation stages of embryogenesis. The two genes are also expressed during the development of the pistillate spikelet and during the first stages of embryo development in adjacent but different tissues. HyPRPmRNA accumulates mainly in the scutellum and HRGP mRNA mainly in the embryo axis and the suspensor. The two genes appear to be under the control of different regulatory pathways during embryogenesis. We show that HyPRP is repressed by abscisic acid and stress treatments, with the exception of cold treatment. In contrast, HRGP is affected positively by specific stress treatments.