Konstantinos Pasentsis

Rolling circle amplification-RACE: a method for simultaneous isolation of 5' and 3' cDNA ends from amplified cDNA templates

Isolation of full-length gene transcripts is important to determine the protein coding region and study gene structure. However, isolation of novel gene sequences is often limited to expressed sequence tags (ESTs) (i.e., short cDNA fragments that predominantly represent the 3′ end of the transcript). Rapid amplification of cDNA ends (RACE) is today by far the most popular approach for obtaining full-length cDNAs when only part of the transcript’s sequence is known.Since its original description (1,2) numerous modifications and improve-ments of the method have been developed and consist of a collection of PCR-based cloning procedures that extend a known cDNA fragment toward the 3′ (3′ RACE) or the 5′ (5′ RACE) cDNA end. The original method is based on attachment of an anchor sequence to one end of the cDNA that can be used as a primer binding template in PCR with a second gene-specific primer from the known part of the gene. Although this procedure seems in theory fast and simple, it is technically difficult and usually requires substantial optimi-zation and several repetitions before satisfactory results can be obtained (3). This is particularly due to the use of a universal primer corresponding to the anchor sequence present in all cDNAs, which may result in a high background of nonspecific products even after a nested PCR with a gene-specific primer internal to the first gene-specific primer is performed. Another drawback of the method is the difficulty of obtaining the full-length 5′ end of the transcript due to the presence of many truncated transcripts in the messenger RNA (mRNA) pool. Several strategies aimed at eliminating these problems have been developed (4–9) and have proven to be very useful in certain applica-tions. One improvement is based on the utilization of a pair of gene-specific primers in inverse PCR on circularized cDNA templates, which would avoid the use of a universal primer and the problems it may generate (4,6,8–10). This strategy also allows the simulta-neous isolation of both cDNA ends in a single reaction (9,10). Some of these procedures require the generation of double-stranded cDNA, including the use of template-switching reverse transcription (9) or a post-reverse transcription adaptor ligation step (10). Methods that are performed directly on first-strand cDNA are complicated by the low efficiency of RNA ligase for the circularization reaction (6) or the need for bridging oligonucleotides for this step (8). Furthermore, existing inverse-RACE methods typically require nested PCR to amplify the transcript of interest, and only a limited number of transcripts can be isolated from a single reverse transcription reaction, making it difficult to analyze rare transcripts from scarce tissue.We describe here an improved inverse-RACE method, which uses CircLigase™ (Epicentre Biotechnologies, Madison, WI, USA) for cDNA circularization, followed by rolling circle amplification (RCA) of the circular cDNA with ϕ29 DNA polymerase (New England Biolabs, Ipswich, MA, USA). In this way, a large amount of the PCR template is produced, allowing the simultaneous isolation of the 3′ and 5′ unknown ends of a virtually unlimited number of transcripts after a single reverse transcription reaction. Figure 1 illus-trates this method, named RCA-RACE. The process takes advantage of the properties of CircLigase to circularize

The maize alternative oxidase 1a (Aox1a) gene is regulated by signals related to oxidative stress.

Abstract We isolated and characterized the expression of Aox1a, a member of the maize alternative oxidase (Aox) small multigene family. Aox1a consists of four exons interrupted by three introns and its promoter harbors diverse stress-specific putative regulatory motifs pointing to complex regulation and response to multiple signals. Responses of Aox1a to such signals were examined and compared with those of maize glutathione S-transferase I (GstI), a typical oxidative stress inducible gene. Potassium cyanide (KCN) and hydrogen peroxide (H2O2) induced a rapid increase of the Aox1a and GstI transcripts, which was persisted in prolonged treatment at high H2O2 concentration only for Aox1a. High concentration of salicylic acid (SA) and salicyl hydroxamic acid (SHAM) induced Aox1a mRNA only after prolonged exposure, while GstI displayed an early strong induction, which declined thereafter. Nitric oxide (NO) induced a high increase of Aox1a after prolonged exposure at high concentration, while GstI displayed a weak response. Our results show that multiple signaling pathways, involved in stress responses, also participate and differentially regulate Aox1a and GstI in maize. A ROS-depended signaling event may be involved, suggesting an essential role of Aox1a under oxidative stress in maize.

Isolation of a differentially spliced C-type flower specific AG-like MADS-box gene from Crocus sativus and characterization of its expression

We have cloned and characterized the expression of Crocus sativus AGAMOUS1 (CsAG1), a putative C-type MADS-box gene homologous to AGAMOUS (AG) from a triploid monocot species crocus (Crocus sativus L.). The typical domain structure of MIKC-type plant MADS proteins was identified. Phylogenetic analysis of the deduced amino acid sequence indicated that the isolated gene forms a clade with the AGAMOUS homologs from the monocots Hyacinthus orientalis and Phalaenopsis equestris. A differential splicing event altering the amino acid sequence at the C terminus was identified, leading to the formation of two mRNAs differing ten nucleotides in size. The presence of both differentially spliced transcripts was restricted only to mature crocus flowers and particularly to stamens and carpels.

Heterotopic expression of B-class floral homeotic genes PISTILLATA/GLOBOSA supports a modified model for crocus (Crocus sativus L.) flower formation

For uncovering and understanding the molecular mechanisms controlling flower development in cultivated Crocus sativus and particularly the transformation of sepals in outer whorl (whorl 1) tepals, we have cloned and characterized the expression of a family of five PISTILLATA/GLOBOSA-like (PI/GLO-like) MADS-box genes expressed in the C. sativus flower. The deduced amino acid sequences of the coded proteins indicated high homology with members of the MADS-box family of transcription factors, and particularly with other members of the PI/GLO family of MADS-box proteins that control floral organ identity. PI/GLO expression studies in cultivated C. sativus uncover the presence of PI/GLO transcripts not only in the second and third whorls of flower organs as expected, but also in the outer whorl tepals that are the sepals in most typical flowers. This heterotopic expression of both B-class genes: PI/GLO and AP3/DEF, known to form heterodimers for stamens and petals (petaloid inner whor l–whorl 2-tepals in C. sativus), explains the homeotic transformation of sepals into outer whorl tepals in this species. Analysis of PI/GLO sequences from C. sativus for putative targets to known micro-RNAs (miRNAs) showed that the target site for ath-miRNA167 found in Arabidopsis thaliana PI is not present in C. sativus, however, the PI/GLO sequences may be regulated by an ath-miRNA163.

Taxonomic Identification of Mediterranean Pines and Their Hybrids Based on the High Resolution Melting (HRM) and trnL Approaches: From Cytoplasmic Inheritance to Timber Tracing

Fast and accurate detection of plant species and their hybrids using molecular tools will facilitate the assessment and monitoring of local biodiversity in an era of climate and environmental change. Herein, we evaluate the utility of the plastid trnL marker for species identification applied to Mediterranean pines (Pinus spp.). Our results indicate that trnL is a very sensitive marker for delimiting species biodiversity. Furthermore, High Resolution Melting (HRM) analysis was exploited as a molecular fingerprint for fast and accurate discrimination of Pinus spp. DNA sequence variants. The trnL approach and the HRM analyses were extended to wood samples of two species (Pinus nigra and Pinus sylvestris) with excellent results, congruent to those obtained using leaf tissue. Both analyses demonstrate that hybrids from the P. brutia (maternal parent) × P. halepensis (paternal parent) cross, exhibit the P. halepensis profile, confirming paternal plastid inheritance in Group Halepensis pines. Our study indicates that a single one-step reaction method and DNA marker are sufficient for the identification of Mediterranean pines, their hybrids and the origin of pine wood. Furthermore, our results underline the potential for certain DNA regions to be used as novel biological information markers combined with existing morphological characters and suggest a relatively reliable and open taxonomic system that can link DNA variation to phenotype-based species or hybrid assignment status and direct taxa identification from recalcitrant tissues such as wood samples.

Sequence Characterization and Expression Analysis of Three APETALA2-like Genes from Saffron Crocus

To further understand flowering and flower organs formation in the monocot crop saffron crocus (Crocus sativus L.), we cloned three APETALA2-like cDNA sequences of the AP2/ERF transcription factor family designated CsatAP2a/b/c as well as the respective promoter region sequences. Bioinformatics analysis with putative orthologous sequences from various plant species suggested that all three cDNA sequences encode for AP2-like proteins with the AP2 characteristic motifs and amino acids. Phylogenetically, the isolated sequences were closest to the AP2-like genes from Pisum sativum, Arabidopsis thaliana and Oryza sativa. Expression analysis indicated that the isolated C. sativus sequences were expressed in all the examined organs. Expression of CsatAP2a/b/c cDNAs was also compared in wild-type and mutant C. sativus flowers lacking stamens or carpels. Sequence analysis of the promoter revealed the presence of putative binding motifis for CCAAT, AP2 and LEAFY transcription factors indicative of regulation by developmental signals.

Isolation, Characterization, and Expression Analysis of an NAP-Like cDNA from Crocus (Crocus sativus L.)

In Arabidopsis, it has been shown that the B-class MADS-box genes are expressed in the developing whorl 2 petals and whorl 3 stamens throughout the ontogeny of these organs. APETALA3/PISTILLATA (AP3/PI) heterodimers act as an inducer of a regulator-coding gene called NAP, a homologue to NAC-like transcription factor genes, required for meristem establishment, separation of floral organs, and leaf senescence. In monocots like crocus (Crocus sativus) cultivated for saffron production, the expression of B-class MADS-box CsatAP3/CsatPI genes extends to whorl 1 where petaloid tepals are formed. We report here for the first time the cloning and characterization of an NAC-like gene, named CsatNAP, from crocus. The sequence alignment indicated that CsatNAP protein contains the typical domain structure of plant NAC proteins consisted of the conserved five subdomains of the N-terminal NAC domain. Phylogenetic analysis revealed that CsatNAP protein falls in the subgroup NAP of the NAC proteins. Expression analysis of crocus NAP indicates that it is expressed in whorl 1, supporting the hypothesis that also in crocus, AP3/PI-like is associated with the expression of Crocus NAP gene. The expression patterns of CsatNAP cDNA were studied in flowers and different flower organs as well as in leaves and different part of leaves during senescence. The CsatNAP transcripts were detected in whole flowers and flower organs. Promoter analysis of CsatNAP revealed a number of putative common cis-regulatory elements and, among them, two CArG boxes indicative of its control by MADS box transcription factors known to bind on CArG sites. Micro-RNA (miRNA) target analysis showed that the NAP sequence of Crocus contain a possible target site for miRNA164. Furthermore, CsatNAP showed increased expression in senescence leaves compared to the green ones.

Isolation of a CENTRORADIALIS/TERMINAL FLOWER1 homolog in saffron (Crocus sativus L.): characterization and expression analysis

Genes in the phosphatidyl-ethanolamine-binding protein (PEBP) family are instrumental in regulating the fate of meristems and flowering time. To investigate the role of these genes in the monocotyledonous plant Crocus (Crocus sativus L), an industrially important crop cultivated for its nutritional and medicinal properties, we have cloned and characterized a CENTRORADIALIS/TERMINAL FLOWER1 (CEN/TFL1) like gene, named CsatCEN/TFL1-like, the first reported CEN/TFL1 gene characterized from such a perennial geophyte. Sequence analysis revealed that CsatCEN/TFL1 shows high similarity to its homologous PEBP family genes CEN/TFL1, FT and MFT from a variety of plant species and maintains the same exon/intron organization. Phylogenetic analysis of the CsatCEN/TFL1 amino acid sequence confirmed that the isolated sequences belong to the CEN/TFL1 clade of the PEBP family. CsatCEN/TFL1 transcripts could be detected in corms, flower and flower organs but not in leaves. An alternative spliced transcript was also detected in the flower. Comparison of expression levels of CsatCEN/TFL1 and its alternative spliced transcript in wild type flower and a double flower mutant showed no significant differences. Overexpression of CsatCEN/TFL1 transcript in Arabidopsis tfl1 plants reversed the phenotype of early flowering and terminal flowering of the tfl1 plants to a normal one. Computational analysis of the obtained promoter sequences revealed, next to common binding motifs in CEN/TFL1-like genes as well as other flowering gene promoters, the presence of two CArG binding sites indicative of control of CEN/TFL1 by MADS-box transcription factors involved in crocus flowering and flower organ formation.

Identification of unknown genetically modified material admixed in conventional cotton seed and development of an event-specific detection method

Entering the second decade of commercialization of biotech crops, the global area cultivated with transgenic plants constantly expands and national legislations in many countries, particularly in the European Union, require identification and labeling of genetically modified material in food and feed. We describe here a procedure for characterizing transgenic material of unknown origin present in conventional seed lots using a genome walking strategy for isolation and characterization of the junction between the inserted transgene construct and the host plant genomic DNA. The procedure was applied to transgenic cotton detected as adventitious or technically unavoidable presence in a conventional commercial cultivar. The structure of the isolated region revealed that the transgenic material derived from Monsantos event 1445 transgenic cotton. Due to the random incorporation of the transgene into the host plants genome, the sequence of the junction region obtained using the genome walking strategy, provided the means to develop an event-specific identification method without prior knowledge for the nature of the transformation event. Thus, we documented a methodology for developing an event-specific detection protocol even without prior knowledge of the genetic modification event.

Cloning and Characterization of FLOWERING LOCUS T -Like Genes from the Perennial Geophyte Saffron Crocus ( Crocus sativus )

The transition to flowering is one of the most important developmental decisions made by plants. At the molecular level, many genes coordinate this transition. Among these, genes encoding for phosphatidylethanolamine-binding proteins (PEBPs) play important roles in regulating flower time and the fate of inflorescence meristem. To investigate the role of PEBPs in an industrially important crop cultivated for its nutritional and medicinal properties, the monocotyledonous species Crocus sativus L., we have isolated three FLOWERING LOCUS T (FT)-like genes designated as CsatFT1-like, CsatFT2-like, and CsatFT3-like. The isolated genes maintain the exon/intron organization of FT-like genes and encode proteins similar to the members of the PEBP family. Phylogenetic and amino acid analysis at critical positions confirmed that the isolated sequence belongs to the FT clade of the PEBP family phylogeny distinctly from the TERMINAL FLOWER 1 (TFL1) and MOTHER OF FT AND TFL1 clades. Expression analysis indicated differences in the expression of the three FT-like genes in different organs and different expressions during the day–night diurnal clock. Additionally, analysis of isolated promoter sequences using computational methods reveals the preservation of common binding motifs in FT-like promoters from other species, thus suggesting their importance among plant species.