José M. Martínez-Zapater

Regulation of flowering time by FVE, a retinoblastoma-associated protein

The initiation of flowering in plants is controlled by environmental and endogenous signals. Molecular analysis of this process in Arabidopsis thaliana indicates that environmental control is exerted through the photoperiod and vernalization pathways, whereas endogenous signals regulate the autonomous and gibberellin pathways. The vernalization and autonomous pathways converge on the negative regulation of FLC, a gene encoding a MADS-box protein that inhibits flowering. We cloned FVE, a component of the autonomous pathway that encodes AtMSI4, a putative retinoblastoma-associated protein. FVE interacted with retinoblastoma protein in immunoprecipitation assays, and FLC chromatin was enriched in acetylated histones in fve mutants. We conclude that FVE participates in a protein complex repressing FLC transcription through a histone deacetylation mechanism. Our data provide genetic evidence of a new developmental function of these conserved proteins and identify a new genetic mechanism in the regulation of flowering.

Low Temperature Induces the Accumulation of Phenylalanine Ammonia-Lyase and Chalcone Synthase mRNAs of Arabidopsis thaliana in a Light-Dependent Manner

Anthocyanins, which accumulate in leaves and stems in response to low temperature and changes in light intensity, are synthesized through the phenylpropanoid pathway that is controlled by key enzymes that include phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS). In this work we demonstrate that PAL and CHS mRNAs accumulate in leaves of Arabidopsis thaliana (L.) Heynh. upon exposure to low temperature in a light-dependent manner. The regulation of the PAL1 gene expression by low temperature and light was examined by analyzing the expression of the [beta]-glucuronidase (uidA) reporter gene in transgenic Arabidopsis plants containing the uidA gene of Escherichia coli under the control of the PAL1 promoter. The results indicate that the accumulation of PAL1 mRNA is transcriptionally regulated. Histochemical staining for [beta]-glucuronidase activity showed that the PAL1 promoter is preferentially activated in photosynthetically active cells, paralleling anthocyanin accumulation. Moreover, we show that light may also be implicated in the regulation of the CHS gene in response to bacterial infiltration. Finally, using two transparent testa Arabidopsis mutants that are unable to accumulate anthocyanins, we demonstrate that these pigments are not required for successful development of freezing tolerance in this species.

Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time

Citrus trees have a long juvenile phase that delays their reproductive development by between 6 and 20 years, depending on the species. With the aim of accelerating their flowering time, we transformed juvenile citrus seedlings to constitutively express the Arabidopsis LEAFY (LFY) or APETALA1 (AP1) genes, which promote flower initiation in Arabidopsis. Both types of transgenic citrus produced fertile flowers and fruits as early as the first year, notably through a mechanism involving an appreciable shortening of their juvenile phase. Furthermore, expression of AP1 was as efficient as LFY in the initiation of flowers, and did not produce any severe developmental abnormality. Both types of transgenic trees flowered in consecutive years, and their flowering response was under environmental control. In addition, zygotic and nucellar derived transgenic seedlings had a very short juvenile phase and flowered in their first spring, demonstrating the stability and inheritance of this trait. These results open new possibilities for domestication, genetic improvement, and experimental research in citrus and other woody species.

A highly repeated DNA sequence in Arabidopsis thaliana

SummaryThree members of a family of highly repeated DNA sequences from Arabidopsis thaliana have been cloned and characterized. The repeat unit has an average length of 180 bp and is tandemly repeated in arrays longer than 50 kb. This family represents more than one percent of the Arabidopsis genome. Sequence comparisons with tandemly repeated DNA sequences from other Cruciferae species show several regions of homology and a similar length of the repeat unit. Homologies are also found to highly repeated sequences from other plant species. When the sequence CCGG occurs in the repeated DNA, the inner cytosine is generally methylated.

Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers

Abstract. AFLP analysis using restriction enzyme isoschizomers that differ in their sensitivity to methylation of their recognition sites has been used to analyse the methylation state of anonymous CCGG sequences in Arabidopsis thaliana. The technique was modified to improve the quality of fingerprints and to visualise larger numbers of scorable fragments. Sequencing of amplified fragments indicated that detection was generally associated with non-methylation of the cytosine to which the isoschizomer is sensitive. Comparison of EcoRI/HpaII and EcoRI/MspI patterns in different ecotypes revealed that 35–43% of CCGG sites were differentially digested by the isoschizomers. Interestingly, the pattern of digestion among different plants belonging to the same ecotype is highly conserved, with the rate of intra-ecotype methylation-sensitive polymorphisms being less than 1%. However, pairwise comparisons of methylation patterns between samples belonging to different ecotypes revealed differences in up to 34% of the methylation-sensitive polymorphisms. The lack of correlation between inter-ecotype similarity matrices based on methylation-insensitive or methylation-sensitive polymorphisms suggests that whatever the mechanisms regulating methylation may be, they are not related to nucleotide sequence variation.

AP2 Gene Determines the Identity of Perianth Organs in Flowers of Arabidopsis thaliana.

We have examined the floral morphology and ontogeny of three mutants of Arabidopsis thaliana, Ap2-5, Ap2-6, and Ap2-7, that exhibit homeotic changes of the perianth organs because of single recessive mutations in the AP2 gene. Homeotic conversions observed are: sepals to carpels in all three mutants, petals to stamens in Ap2-5, and petals to carpels in Ap2-6. Our analysis of these mutants suggests that the AP2 gene is required early in floral development to direct primordia of the first and second whorls to develop as perianth rather than as reproductive organs. In addition, our results support one of the two conflicting hypotheses concerning the structures of the calyx and the gynoecium in the Brassicaceae.

Different roles of flowering-time genes in the activation of floral initiation genes in Arabidopsis.

We have analyzed double mutants that combine late-flowering mutations at four flowering-time loci (FVE, FPA, FWA, and FT) with mutations at the LEAFY (LFY), APETALA1 (AP1), and TERMINAL FLOWER1 (TFL1) loci involved in the floral initiation process (FLIP). Double mutants between ft-1 or fwa-1 and lfy-6 completely lack flowerlike structures, indicating that both FWA and FT act redundantly with LFY to control AP1. Moreover, the phenotypes of ft-1 ap1-1 and fwa-1 ap1-1 double mutants are reminiscent of the phenotype of ap1-1 cal-1 double mutants, suggesting that FWA and FT could also be involved in the control of other FLIP genes. Such extreme phenotypes were not observed in double mutants between fve-2 or fpa-1 and lfy-6 ap1-1. Each of these showed a phenotype similar to that of ap1-1 or lfy-6 mutants grown under noninductive photoperiods, suggesting a redundant interaction with FLIP genes. Finally, the phenotype of double mutants combining the late-flowering mutations with tfl1-2 were also consistent with the different roles of flowering-time genes.

High throughput SNP discovery and genotyping in grapevine (Vitis vinifera L.) by combining a re-sequencing approach and SNPlex technology

BackgroundSingle-nucleotide polymorphisms (SNPs) are the most abundant type of DNA sequence polymorphisms. Their higher availability and stability when compared to simple sequence repeats (SSRs) provide enhanced possibilities for genetic and breeding applications such as cultivar identification, construction of genetic maps, the assessment of genetic diversity, the detection of genotype/phenotype associations, or marker-assisted breeding. In addition, the efficiency of these activities can be improved thanks to the ease with which SNP genotyping can be automated. Expressed sequence tags (EST) sequencing projects in grapevine are allowing for the in silico detection of multiple putative sequence polymorphisms within and among a reduced number of cultivars. In parallel, the sequence of the grapevine cultivar Pinot Noir is also providing thousands of polymorphisms present in this highly heterozygous genome. Still the general application of those SNPs requires further validation since their use could be restricted to those specific genotypes.ResultsIn order to develop a large SNP set of wide application in grapevine we followed a systematic re-sequencing approach in a group of 11 grape genotypes corresponding to ancient unrelated cultivars as well as wild plants. Using this approach, we have sequenced 230 gene fragments, what represents the analysis of over 1 Mb of grape DNA sequence. This analysis has allowed the discovery of 1573 SNPs with an average of one SNP every 64 bp (one SNP every 47 bp in non-coding regions and every 69 bp in coding regions). Nucleotide diversity in grape (π = 0.0051) was found to be similar to values observed in highly polymorphic plant species such as maize. The average number of haplotypes per gene sequence was estimated as six, with three haplotypes representing over 83% of the analyzed sequences. Short-range linkage disequilibrium (LD) studies within the analyzed sequences indicate the existence of a rapid decay of LD within the selected grapevine genotypes. To validate the use of the detected polymorphisms in genetic mapping, cultivar identification and genetic diversity studies we have used the SNPlex™ genotyping technology in a sample of grapevine genotypes and segregating progenies.ConclusionThese results provide accurate values for nucleotide diversity in coding sequences and a first estimate of short-range LD in grapevine. Using SNPlex™ genotyping we have shown the application of a set of discovered SNPs as molecular markers for cultivar identification, linkage mapping and genetic diversity studies. Thus, the combination a highly efficient re-sequencing approach and the SNPlex™ high throughput genotyping technology provide a powerful tool for grapevine genetic analysis.

Application of AFLPs to the characterization of grapevine Vitis vinifera L. genetic resources. A case study with accessions from Rioja (Spain)

Abstract AFLPs were used to characterize 67 different grapevine accessions from a collection of D.O.Ca. Rioja in Spain. A correct selection of primers and selective nucleotides allowed us to maximize the number of amplified fragments analyzed per reaction yielding an average of 100 per reaction, 49% of which were polymorphic. Based on the presence or absence of amplified fragments for each genotype resulting from a reaction with two primer combinations, we have established the genetic similarity between the different accessions in the collection. These results allowed us to resolve different genotypes maintained under the same name (homonyms) and to identify the same genotype under different names (synonyms) thus permitting the elimination of redundant germplasm. Furthermore, by providing information on more than 50 polymorphic loci per reaction, a few reactions were sufficient to identify distinct AFLP patterns characteristic of specific clones, with different agronomic and organoleptic features, belonging to the same cultivar. The possibility for clonal identification, shown here for grapevines, can have important implications in the protection and management of clonal selections.

Mutations in the Ca2+/H+ Transporter CAX1 Increase CBF/DREB1 Expression and the Cold-Acclimation Response in Arabidopsis

Transient increases in cytosolic free calcium concentration ([Ca2+]cyt) are essential for plant responses to a variety of environmental stimuli, including low temperature. Subsequent reestablishment of [Ca2+]cyt to resting levels by Ca2+ pumps and C-REPEAT BINDING FACTOR/DEHYDRATION RESPONSIVE ELEMENT BINDING FACTOR 1 (Ca2+/H+) antiporters is required for the correct transduction of the signal. We have isolated a cDNA from Arabidopsis that corresponds to a new cold-inducible gene, RARE COLD INDUCIBLE4 (RCI4), which was identical to CALCIUM EXCHANGER 1 (CAX1), a gene that encodes a vacuolar Ca2+/H+ antiporter involved in the regulation of intracellular Ca2+ levels. The expression of CAX1 was induced in response to low temperature through an abscisic acid–independent pathway. To determine the function of CAX1 in Arabidopsis stress tolerance, we identified two T-DNA insertion mutants, cax1-3 and cax1-4, that display reduced tonoplast Ca2+/H+ antiport activity. The mutants showed no significant differences with respect to the wild type when analyzed for dehydration, high-salt, chilling, or constitutive freezing tolerance. However, they exhibited increased freezing tolerance after cold acclimation, demonstrating that CAX1 plays an important role in this adaptive response. This phenotype correlates with the enhanced expression of CBF/DREB1 genes and their corresponding targets in response to low temperature. Our results indicate that CAX1 ensures the accurate development of the cold-acclimation response in Arabidopsis by controlling the induction of CBF/DREB1 and downstream genes.