Apostolos Kalivas

Abstract: The actin-binding protein profilin (PRF) plays an important role in cell growth and expansion by regulating the organization of the actin filaments. Recent studies have reported association between fiber elongation in cultivated cotton (Gossypium hirsutum) and PRF expression. In the present study, we cloned four genomic clones from allotetraploid cotton (G. hirsutum) and its putative diploid progenitors (G. arboreum and G. raimondii) designated GhPRF1_A, GhPRF1_D, GaPRF1, and GrPRF1 encoding cotton PRF and characterized their genomic structure, phylogenetic relationships and promoter structure. Sequence analysis of the coding regions of all clones resulted in a single protein product which revealed more than 80% similarity to most plant PRFs and a typical organization with an actin-binding and a polybasic phospholipid binding motif at the carboxy terminus. DNA blot hybridization suggested that PRF gene is present with more than one copy in the allotetraploid species G. hirsutum. Expression analysis performed in various organs of cultivated cotton revealed that the PRF gene was preferentially expressed in cotton fibers. Very low levels of expression were observed in whole flowers, while PRF transcripts were not detected in other organs examined. Furthermore, higher levels of expression were observed at the early stages of cotton fiber development (at 10 days post anthesis), indicative that this gene may play a major role in the early stages of cotton fiber development. Quantitation of the expression by real-time PCR revealed higher expression levels in a G. hirsutum variety with higher fiber percentage compared to a variety with lower percentage. In addition, higher levels of expression were found in cultivated allotetraploid G. barbadense cotton species with higher fiber length in comparison to cultivated allotetraploid G. hirsutum.

Abstract: To further understand flowering and flower organ formation in the monocot crop saffron crocus (Crocus sativus L.), we cloned four MIKC(c) type II MADS-box cDNA sequences of the E-class SEPALLATA3 (SEP3) subfamily designated CsatSEP3a/b/c/c_as as well as the three respective genomic sequences. Sequence analysis showed that cDNA sequences of CsatSEP3 c and c_as are the products of alternative splicing of the CsatSEP3c gene. Bioinformatics analysis with putative orthologous sequences from various plant species suggested that all four cDNA sequences encode for SEP3-like proteins with characteristic motifs and amino acids, and highlighted intriguing sequence features. Phylogenetically, the isolated sequences were closest to the SEP3-like genes from monocots such as Asparagus virgatus, Oryza sativa, Zea mays, and the dicot Arabidopsis SEP3 gene. All four isolated C. sativus sequences were strongly expressed in flowers and in all flower organs: whorl1 tepals, whorl2 tepals, stamens and carpels, but not in leaves. Expression of CsatSEP3a/b/c/c_as cDNAs was compared in wild-type and mutant flowers. Expression of the isolatedCsatSEP3-like genes in whorl1 tepals together with E-class CsatAP1/FUL subfamily and B-class CsatAP3 and CsatPI subfamilies of genes, fits the ABCE "quartet model," an extended form of the original ABC model proposed to explain the homeotic transformation of whorl1 sepals into whorl1 tepals in Liliales and Asparagales plants such as C. sativus. This conclusion was also supported by the interaction of the CsatSEP3b protein with CsatAP1/FUL and CsatAP3 proteins. In contrast, expression of both B-class CsatAP3 and CsatPI genes and the C-class CsatAGAMOUS genes together with E-class CsatSEP3-like genes in carpels, without any phenotypic effects on carpels, raises questions about the role of these gene classes in carpel formation in this non-grass monocot and requires further experimentation. Finally, taking advantage of the size and sequence differences in amplified genomic sequences of the triploid C. sativus and comparing them with the respective sequences from C. tomasii, C. hadriaticus and C. cartwrightianus, three putative wild-type diploid progenitor species, we examined the origin of CsatSEP3a sequence.

Abstract: Cotton is the most important textile plant in the world and is one of the most important crops for the production of oilseed. Because of its worldwide economic importance, new cultivars are constantly being released in the world and consequently in the Greek market, as Greece is the largest producer in Europe. We used simple sequence repeat (SSR) markers for the identification and the phylogenetic analysis of the most widely cultivated cotton cultivars in Greece. Initially, we used 12 pairs of SSR molecular markers for the analysis of 29 cultivars of Gossypium hirsutum and an interspecific hybrid (G. hirsutum x G. barbadense). Of the 12 pairs of SSR primers, 11 amplified polymorphic products, while one pair did not amplify any product. Globally, 17 polymorphic marker loci were identified. Two to four different alleles were amplified at each genomic locus, with a mean of 2.53 alleles per locus. Among the 30 genotypes that we analyzed, the polymorphism information content ranged from 0 to 0.548, with a mean of 0.293. Three main groups were formed among the 30 genotypes when a phylogenetic analysis was performed using UPGMA. Computational analysis of each molecular marker separately showed an association of SSR markers with agronomic traits such as fiber quality. To our knowledge, this is the first in-depth molecular analysis of cotton cultivars grown in Greece using SSR markers. An analysis of association of SSR markers with fiber quality traits of 29 cotton cultivars is reported for the first time.

Abstract: 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.

Abstract: We describe an improvement of the RCA-RACE (rolling circle amplification-rapid amplification of cDNA ends) method, called family RCA-RACE (famRCA-RACE). The method is based on the generation of circular cDNA fragments, followed by rolling circle amplification of the circular cDNA using phi29 DNA polymerase and the application of PCR using degenerate outworking primers, designed for a conserved region of homologous genes, that allows the isolation of homologous cDNA sequences expressed in the mRNA preparation in a single polymerase chain reaction (PCR). As an example we present the isolation of seven NAC-like transcription factors cDNA sequences expressed in Crocus sativus flower, used for saffron production. Sequence alignment revealed that CsatNAC proteins contain the typical domain structure of plant NAC proteins, consisting of the conserved N-terminal NAC domain used to design the primers and the five subdomains. Phylogenetic analysis revealed that CsatNAC proteins fall in subgroup I of the NAC family of proteins.

Abstract: Zygote arrest 1 (Zar1) is an oocyte-specific maternal effect gene that plays essential role during the oocyte-to-embryo transition in many species including human, mouse, rat, bovine, pig, frog and zebrafish. This study reports that this maternal-effect gene is also conserved in the avian species, namely the chicken Gallus gallus domesticus. In silico mining of the chicken genome shows that the chicken Zar1 (cZar1) gene consists of four exons and three introns, and the coding region of this gene is 903 bp. Using rapid amplification of cDNA ends analysis, the 3β€²-untranslated regions of the gene were isolated, where putative Pumilio (XPum) recognition sites as well as hexamer polyadenylation signals were identified. The gene is predicted to encode a 33 kDa protein, characterized by a conserved C-terminal FYVE/PHD zinc finger domain. In the present study the expression profile of this gene in chicken tissues and embryos was analyzed using reverse transcription-polymerase chain reaction (RT-PCR). The data presented in this study suggests that Zar1 is conserved in another vertebrate species, the chicken, and is preferentially expressed in the chicken reproductive tract, oocytes and embryos, suggesting a role in reproduction and embryonic development.

Abstract: Crocus (Crocus sativus L.) is a crop species cultivated for its flowers and, more specifically, for its red stigmas. The flower of crocus is bisexual and sterile, since crocus is a triploid species. Its perianth consists of six petaloid tepals: three tepals in whorl 1 (outer tepals) and three tepals in whorl 2 (inner tepals). The androecium consists of three distinct stamens and the gynoecium consists of a single compound pistil with three carpels, a single three-branched style, and an inferior ovary. The dry form of the stigmas constitutes the commercial saffron used as a food additive, in the coloring industry, and in medicine. In order to uncover and understand the molecular mechanisms controlling flower development in cultivated crocus and its relative wild progenitor species, and characterize a number of crocus flower mutants, we have cloned and characterized different, full-length, cDNA sequences encoding MADS-box transcription factor proteins involved in flower formation. Here we review the different methods followed or developed for obtaining these sequences involving conventional 5 inverted exclamation markä 3 inverted exclamation markä RACE, as well as newly developed methods from our group, named Rolling Circle Amplification C RACE (RCA-RACE) and its modification named familyRCA-RACE (famRCA-RACE). Furthermore, the characteristics of the protein structure and their common and specific domains for each type of MADS-box transcription factors in this lower nongrass monocot belonging to the Iridaceae family are described. Finally, a phylogenetic tree of all the MADS-box sequences available in our lab is presented and discussed in relation to other data from studies of species of the Iridaceae group and closely related families from an evolutionary perspective. The structural and phylogenetic analyses are based on both published and unpublished data.

Abstract: 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.

Abstract: We have cloned and characterized the expression of two flower specific B-class APETALA3-like (AP3-like) MADS-box sequences of cultivated crocus (Crocus sativus L). Based on sequencing data two sequences designated CsatAP3a and CsatAP3b could be distinguished. These were different in the 5' and 3' untranslated regions and had five single nucleotide differences in the coding region that led to a single amino acid difference in the coded protein. The deduced amino acid sequences of the genes indicated high similarity with members of the MADS-box family of transcription factors, and particularly with other members of the paleo-AP3 lineage of B-class MADS-box proteins that control floral organ identity. Phylogenetic analysis at the amino acid level confirmed that the isolated sequences belong to the monocot-specific paleoAP3 clade. In the sequence the typical domain structure of plant MADS box proteins was observed. The conserved N-terminal MADS-box, the I domain, the central K domain and a C terminal domain harboring a paleoAP3 motif were identified. Expression analysis indicated that transcripts of CsatAP3 and also an isolated PISTILLATA-like CsatPIc sequence are not restricted to organs of the second and third whorls of the flowers but are present also in the tepals of the first whorl and stigmata of the mature crocus flower of the fourth whorl. Extension of CsatAP3 and CsatPIc expression in organs of the first whorl could be a supportive evidence to explain the homeotic transformation of sepals into tepals in crocus. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Abstract: Flowering and flower formation is a major developmental switch in plants where different genes are expressed in a coordinate manner for reproductive success. In order to uncover and understand the molecular mechanisms controlling flowering and flower formation in cultivated Crocus sativus L. and to characterize a number of crocus flower mutants, we have cloned and characterized different cDNA and genomic sequences encoding MADS-box transcription factor proteins involved in flowering and flower formation using a combination of different methods including conventional 5' and 3' RACE, as well as newly developed methods to isolate full cDNAs and genomic sequences based on Rolling Cycle Amplification (RCA).

Abstract: Flowering and flower formation is a major developmental switch in plants where different genes are expressed in a coordinate manner for reproductive success. Among these genes MADS domain transcription factors and FLOWER LOCUS T like genes play important roles in various developmental processes in flowering plants, from transition to flowering to the specification of floral organ identity. For uncovering and understanding the molecular mechanisms controlling flowering, flower development and flower formation in cultivated Crocus sativus L., we present here the isolation and characterization of a series of flowering genes. Furthermore, based on the isolated genomic sequences of these genes, we have developed and used three SSR molecular markers located at the 5'-UTR of isolated MADS box genes in order to analyze 11 Crocus accessions from different countries.