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Generation of expressed sequence tags from carob (Ceratonia siliqua L.) flowers for gene identification and marker development
Abstract
Carob (Ceratonia siliqua L.) is a caesalpinoid legume tree showing labile sex expression. With the main aims of identifying flower-expressed genes
and of developing specific markers, 1,056 clones from a complementary DNA library of carob flowers were bidirectionally sequenced.
A total of 1,377 high-quality expressed sequence tags were clustered into 1,096 unigenes. Basic Local Alignment Search Tool
and Gene Ontology functional annotation allowed to identify several agronomically important genes, such as those involved
in flower development and sexual reproduction, response to stress, galactomannan synthesis, and hormone pathways. Genes involved
in the ethylene biosynthesis and response were quantified in developing flowers of three sex genotypes (male, female, and
hermaphrodite) using quantitative reverse transcription polymerase chain reaction. The transcript levels of 1-aminocyclopropane-1-carboxylic
acid oxidase, acting downstream in ethylene pathway, and Ethylene Insensitive 3 (EIN3)-like, a transcription factor involved
in ethylene signaling, were directly correlated with maleness, indicating a possible role of ethylene in carob sex expression.
Furthermore, the first set of carob genic microsatellites was developed, which might be useful for genotyping and genetic
diversity analysis.
... Carob is an evergreen, dioecious tree and it is a constituent of the Mediterranean thermophilus woodlands vegetation (Zohary 2002). Carob is widespread in all Mediterranean countries in different pedoclimatic environments, making this plant one of the most characteristic of the coastal Mediterranean landscape in which it is particularly common in marginal areas due to its lower agronomic requirements compared to other fruit species (Batlle and Tous 1997;Caruso et al. 2008a;Tous et al. 2013). ...
... Currently, there are only a few studies investigating the level of genetic diversity existing in carob tree through molecular markers, while to the best of our knowledge, no studies focused on marker-trait association nor on the setup of a core collection. RAPD (Random Amplified Polymorphic D N A ) a n d A F L P ( A m p l i f i ed F r a g m e n t L e n g t h Polymorphism) markers have first been used to highlight the genetic diversity within carob trees (Talhouk et al. 2005;Konate et al. 2007;Caruso et al. 2008a). The first set of carob tree short sequence repeat (SSR) marker was developed by Caruso et al. (2008b) from floral gene sequences (Expressed Sequence Tags, ESTs). ...
... Genotyping was performed through the analysis of eight nuclear SSR markers and the sequencing of a chloroplastic locus. The eight SSRs used in this study were selected on the base of their performances from a set of 11 EST-SSRs developed by Caruso et al. (2008a). The primer sequences of the SSRs were retrieved from the same paper. ...
Carob (Ceratonia siliqua L.) is an important evergreen tree of the Mediterranean landscape. Its economic interest is increasing thanks also to the presence, in the seeds, of the locust bean gum (LBG), a galactomannan largely used by the food industry as a stabilizer and thickening agent. Its economic and ecological values make the understanding of carob genetic diversity of great interest both for breeding and conservation purposes. The world’s largest carob germplasm collection was genotyped using both eight carob-specific nuclear short sequence repeat (nSSR) and the sequencing of a chloroplast locus. The collection is composed of 215 accessions introduced from 12 countries of origin spanning from traditional to novel areas of cultivation. To assess the genetic diversity of the collection, several approaches were coupled: structure analysis, principal component analysis (PCA), and graphic clustering either from dissimilarity data and coancestry data. Structure analysis suggested the presence of two distinct genetic pools: one characterizing northeastern Spain and the second spread in other countries and southern Spain. The PCA and discriminant analysis of principal component (DAPC) complemented the structure results allowing a better understanding of the genetic differences between countries while the network-joining analysis provided additional insights on the similarity between individuals. Short sequence repeat (SSR) data coupled with phenotypic data (floral sex and status) were also used to define the first core collection of carobs. Multi-approach analysis of genetic diversity together with the definition of a core collection represent useful tools for the setup of genetic-guided intervention both for conservation and breeding purposes.
... Also, Caruso et al. (2008a) used AFLP markers to estimate intra-specific genetic diversity among 41 carob accession from Italy, Spain, Turkey and Israel. Recently Caruso et al. (2008b) developed the first set of carob microsatellites (or SSR, Simple Sequence Repeat) from floral gene sequences (Expressed Sequence Tags, ESTs), however these markers have never been used for genetic diversity studies. The aim of this work was to test the usefulness of species-specific microsatellite markers for the genotyping of carob tree accessions from Italy, Malta and Spain. ...
... A set of twenty EST-SSRs (Table 2) isolated from carob (Caruso et al., 2008b) was used for molecular fingerprinting. Such markers were tested initially on a small number of genotypes in order to verify the performance of amplification. ...
Carob is a caesalpinoid tree species widespread in the Mediterranean. In this paper, carob EST-SSR (Expressed Sequence Tags-Simple Sequence Repeat) markers were used to assess the level of genetic diversity among 71 cultivars and accessions collected in three Mediterranean countries (Italy, Malta and Spain). Starting from 20 microsatellite loci, we identified a set of 9 polymorphic EST-SSRs, with a number of alleles per locus ranging from 2 to 7, and a number of genotypes from 3 to 13. The nine polymorphic EST-SSRs discriminated most of the analyzed genotypes with the exceptions of some cultivated clones having similar phenotypic features and probably belonging to cultivar-populations, and clarified some cases of homonymy.
A neighbor joining dendrogram generated three main clusters which did not show a correlation with the distribution areas of the analyzed genotypes. A Bayesian analysis evidenced a high degree of admixture among the gene pools from the three countries. The low level of diversification among geographical areas reveals that the asexual propagation of selected clones played a major role in the diffusion of the species in the western Mediterranean. The work described in this paper represents the first report of carob genotyping achieved through microsatellite markers.
... Further complementary studies involving reproduction biology characteristics (Caruso et al., 2008), chemical attributes (Dakia et al., 2007) and genetic analysis (La Malfa et al., 2014) are necessary to investigate the performance and real value of this heritage, prior to conserving and utilizing the germplasm in pre-breeding and breeding programs. Table 2. Leaf characteristics of 59 centennial carob trees growing in Lebanon. ...
This study aimed to assess the ancient carob germplasm growing in Lebanon. A set of 59 old trees sampled from different locations across the country (between 0 and 554 m a.s.l.) were subject to morphological characterization by using 33 traits related to the tree, the leaf, the pod and the seed. Results revealed important morphological variability within the Lebanese carob germplasm. The most variable traits were the number of aborted seeds per pod, seed yield, pulp weight, pod weight and pod volume. Correlation analysis showed a negative correlation of both pod size and weight with seed yield, and a positive correlation between pod length and total seed weight. Evaluation of carob morphological characters and eco-geographic parameters revealed positive correlation of seed length, chord length and pod width with the longitude, in addition to a slight positive correlation of pod weight with latitude and longitude. Principal component analysis allowed extracting the most active and discrimi-nant morphological variables, which were mainly represented by pod weight, width, length, thickness and shape, chord length, and individual seed weight and length. Cluster analysis revealed a clear differentiation between two main morphological groups; the first cluster was characterized by long and heavy pods and seeds, while the second cluster was distinguished by medium to small pods and seeds. Two eco-geographic sub-clusters could be differentiated, Mount Lebanon and the South, while the trees of Beirut and the North were dispersed in various subgroups. According to these results, the Lebanese carob germplasm might be a reservoir of genetic diversity that should be further investigated by complementary studies including flowering characteristics, pod chemical quality attributes and genetic analysis.
... Further complementary studies involving reproduction biology characteristics (Caruso et al., 2008), chemical attributes (Dakia et al., 2007) and genetic analysis (La Malfa et al., 2014) are necessary to investigate the performance and real value of this heritage, prior to conserving and utilizing the germplasm in pre-breeding and breeding programs. Supplementary Table 4. Pod characteristics of 52 centennial carob trees (the non-productive trees considered as male were excluded). ...
This study aimed to assess the ancient carob germplasm growing in Lebanon. A set of 59 old trees sampled from different locations across the country (between 0 and 554 m a.s.l.) were subject to morphological characterization by using 33 traits related to the tree, the leaf, the pod and the seed. Results revealed important morphological variability within the Lebanese carob germplasm. The most variable traits were the number of aborted seeds per pod, seed yield, pulp weight, pod weight and pod volume. Correlation analysis showed a negative correlation of both pod size and weight with seed yield, and a positive correlation between pod length and total seed weight. Evaluation of carob morphological characters and eco-geographic parameters revealed positive correlation of seed length, chord length and pod width with the longitude, in addition to a slight positive correlation of pod weight with latitude and longitude. Principal component analysis allowed extracting the most active and discriminant morphological variables, which were mainly represented by pod weight, width, length, thickness and shape, chord length, and individual seed weight and length. Cluster analysis revealed a clear differentiation between two main morphological groups; the first cluster was characterized by long and heavy pods and seeds, while the second cluster was distinguished by medium to small pods and seeds. Two eco-geographic sub-clusters could be differentiated, Mount Lebanon and the South, while the trees of Beirut and the North were dispersed in various subgroups. According to these results, the Lebanese carob germplasm might be a reservoir of genetic diversity that should be further investigated by complementary studies including flowering characteristics, pod chemical quality attributes and genetic analysis. © 2018 Journal of the American Pomological Society. All rights reserved.
... Further complementary studies involving reproduction biology characteristics (Caruso et al., 2008), chemical attributes (Dakia et al., 2007) and genetic analysis (La Malfa et al., 2014) are necessary to investigate the performance and real value of this heritage, prior to conserving and utilizing the germplasm in pre-breeding and breeding programs. caRoB ...
Pollen source, seed set and subsequent seed development are necessary prerequisites for apple fruit production. Pollinizer genotype in particular can have a remarkable impact on fertilization and therefore seed set. However, there is little information published on the most effective and compatible pollinizers for particular commercial cultivars. This study was conducted to determine the effect of three pollen sources, crabapple ('Ralph Shay' in 2013 and Malus floribunda in 2014), 'Delicious', and 'Golden Delicious' on seed number, fruit quality and subsequent return bloom of 'Honeycrisp', 'Fuji' and 'Gala' apples. The effects of 'Gala' pollinizing 'Honeycrisp' trees were also investigated. There was no effect of pollen source on fruit fresh weight, soluble solids concentration or starch pattern index. Seed number per fruit and seed fresh weight per fruit were significantly influenced by pollen source. When 'Ralph Shay' or Malus floribunda crabapples were used as pollinizers, fruit contained fewer seeds and lower seed fresh weight compared with 'Delicious', 'Golden Delicious' and 'Gala' pollinizers; however, the trend was not statistically significant for all cultivars and years. Fruit fresh weight increased linearly with seed number. Pollen source had no influence on return bloom regardless of female cultivar or year. Return bloom was negatively related to fruit fresh weight and seed number per fruit. These results indicate that pollen source and seed number per fruit influence fruit set, fruit quality, biennial bearing potential of 'Honeycrisp', and therefore should be factors that are considered in the orchard design process. Based on our findings, we recommend growers to do not plant 'Ralph Shay' or Malus floribunda crabapples as pollinizers for ?Honeycrisp'. © 2018 Journal of the American Pomological Society. All rights reserved.
... Gene expression studies indicate the involvement of RCD1 orthologs in stress and developmental responses in other species than Arabidopsis: In Salix, a cDNA encoding a protein similar to the closest homolog of RCD1 in Arabidopsis, SRO1 (see chapter 8.3), was regulated in response to chromium treatment (Quaggiotti et al. 2007). A role for an RCD1 ortholog in Ceratonia siliqua was hypothesized in organ abortion or ethylene signaling during floral development (Caruso et al. 2008) and an RCD1-like gene was up-regulated in response to heat shock-induced chilling resistance in Citrus fruits (Sanchez-Ballesta et al. 2003). In an elevated carbon dioxide experiment in Populus, a WWE domain containing protein-coding cDNA, a likely RCD1 ortholog, was downregulated by the carbon dioxide enrichment (Taylor et al. 2005). ...
Plants constantly face adverse environmental conditions, such as drought or extreme temperatures that threaten their survival. They demonstrate astonishing metabolic flexibility in overcoming these challenges and one of the key responses to stresses is changes in gene expression leading to alterations in cellular functions. This is brought about by an intricate network of transcription factors and associated regulatory proteins. Protein-protein interactions and post-translational modifications are important steps in this control system along with carefully regulated degradation of signaling proteins. This work concentrates on the RADICAL-INDUCED CELL DEATH1 (RCD1) protein which is an important regulator of abiotic stress-related and developmental responses in Arabidopsis thaliana. Plants lacking this protein function display pleiotropic phenotypes including sensitivity to apoplastic reactive oxygen species (ROS) and salt, ultraviolet B (UV-B) and paraquat tolerance, early flowering and senescence. Additionally, the mutant plants overproduce nitric oxide, have alterations in their responses to several plant hormones and perturbations in gene expression profiles. The RCD1 gene is transcriptionally unresponsive to environmental signals and the regulation of the protein function is likely to happen post-translationally. RCD1 belongs to a small protein family and, together with its closest homolog SRO1, contains three distinguishable domains: In the N-terminus, there is a WWE domain followed by a poly(ADP-ribose) polymerase-like domain which, despite sequence conservation, does not seem to be functional. The C-terminus of RCD1 contains a novel domain called RST. It is present in RCD1-like proteins throughout the plant kingdom and is able to mediate physical interactions with multiple transcription factors. In conclusion, RCD1 is a key point of signal integration that links ROS-mediated cues to transcriptional regulation by yet unidentified means, which are likely to include post-translational mechanisms. The identification of RCD1-interacting transcription factors, most of whose functions are still unknown, opens new avenues for studies on plant stress as well as developmental responses. Kasvit taiteilevat jatkuvasti erilaisten haitallisten ympäristötekijöiden ristitulessa. Näitä tekijöitä, kuten korkeita tai matalia lämpötiloja, kuivuutta ja taudinaiheuttajia, vastaan kasvit käyvät eräänlaista asemasotaa: Ne eivät kykene etsiytymään miellyttävämpään ympäristöön, vaan niiden ainoat aseet ovat äärimmäisen joustava aineenvaihdunta sekä tarkoin säädelty puolustukseen liittyvien geenien aktivoiminen. Kasvisoluissa onkin joukoittain geeniekspression säätelytekijöitä, eli transkriptiotekijöitä, sekä niihin liittyviä apuproteiineja, joiden toimintaa säädellään tarkoin kohdennetuilla proteiinien välisillä interaktioilla, proteiinien pintarakenteiden joustavalla muokkaamisella ja niiden tarkoituksellisella hajoittamisella. Tässä työssä on tutkittu lituruohon RADICAL-INDUCED CELL DEATH1-proteiinia (RCD1), jonka tiedetään olevan sekä stressi- että kehitysvasteiden tärkeä säätelijä. Tämän proteiinin toiminnan häiriintyminen johtaa moninaisiin ilmiasuihin, kuten otsoni- ja suolaherkkyyteen sekä parantuneeseen UV-säteilyn kestoon, minkä lisäksi kasviyksilöiden elinkierto on nopeutunut ja niiden lehtien kehityksessä on muutoksia. Koko genomin kattavissa ekspressiotutkimuksissa nämä ilmiöt ovat myös nähtävissä suurina muutoksina geenien ilmenemisessä. Tässä työssä on osoitettu, että RCD1 sisältää tähän asti tuntemattoman proteiinidomeenin, jonka avulla se pystyy olemaan kosketuksissa moniin erilaisiin transkriptiotekijöihin ja että proteiinin muut osat osallistuvat todennäköisesti näiden tekijöiden toiminnan säätelyyn. Lisäksi lituruohon genomista löytyi viisi RCD1:n sukulaisproteiinia, joiden toiminta on toistaiseksi hämärän peitossa. Tämä proteiiniperheen osoitettiin löytyvän myös kaikkien tutkittujen maakasvien genomeista, mikä viittaa siihen, että niiden toiminta on olennaista kaikkien näiden kasvien toiminnalle. RCD1:n eikä sen sukulaisporteiinien biokemiallista toimintaa tunneta. On tärkeää selvittää mekaanisella tasolla, miten nämä proteiinit muokkaavat transkriptiotekijöitä vai muodostavatko ne esimerkiksi jonkin säätelyyn tarvittavan proteiinikompleksin rungon, jossa muut proteiiinit hoitavat varsinaisen biokemiallisen muokkauksen. Tässä työssä on siis löydetty uusi transkriptiotekijöiden kontrollointijärjestelmä, jonka tunteminen tulee valaisemaan huomattavasti sekä kasvien stressi- että kehityssignalointia. Tämä puolestaan on osa kasvien kokonaisvaltaista toiminnan ymmärtämistä, joka on edellytyksenä kasvien rationaaliselle jalostamiselle ja vastaamiselle muuttuvan ympäristön maataloudelle asettamiin haasteisiin.
The carob tree, Ceratonia siliqua L, is a characteristic constituent of the evergreen, "maquis" and "garigue" vegetation type in low-altitude areas in the Mediterranean Basin. All over these territories, this dioecious and thermophilous tree has been extensively cultivated for its pods. These trees are used for both human consumption and as a sugar-rich animal feed. However, currently the main interest is seed production for gum extraction from the endosperm, which is used as a stabilizer in numerous commercial food products. Most cultivars are female, and require the presence of pollen donors in the orchards. The carob is mainly insect-pollinated (flies, bees, and wasps), but is also wind-pollinated, depending on the habitat. The carob tree shows some outstanding features, like rusticity and drought resistance, and produces under reduced orchard management, and thus is well suited for part-time farming. Carob's main limitation is its frost sensitivity. In addition, modern carob orchards start bearing earlier (fourth year after budding) than traditional carob plots, and they increase yield steadily in response to minimum cultural care and deficit irrigation. This crop has received little attention until now, but currently it is being reemphasized as an alternative in dryland (500mm) or in supplementary drip irrigation areas with a Mediterranean climate for diversification and revitalization of coastal agriculture. This chapter describes carob's economic importance worldwide and their genetic resources and it also reviews various aspects of its taxonomy, botany, origin, ecology, properties, uses, diversity, and breeding. All the life cycle descriptions are referred to the northern hemisphere climatic conditions. In addition, a full account of the crop-producing areas, agronomy, limitations, industry market, and prospects is presented.
Cassava (Manihot esculenta Crantz) is a staple food for over 600 million people in the tropics and subtropics and is increasingly used as an industrial crop for starch production. Cassava has a high growth rate under optimal conditions but also performs well in drought-prone areas and on marginal soils. To increase the tools for understanding and manipulating drought tolerance in cassava, we generated expressed sequence tags (ESTs) from normalized cDNA libraries prepared from dehydration-stressed and control well-watered tissues. Analysis of a total of 18,166 ESTs resulted in the identification of 8,577 unique gene clusters (5,383 singletons and 3,194 clusters). Functional categories could be assigned to 63% of the unigenes, while another similar to 11% were homologous to hypothetical genes with unclear functions. The remaining similar to 26% were not significantly homologous to sequences in public databases suggesting that some may be novel and putatively specific to cassava. The dehydration-stressed library uncovered numerous ESTs with recognized roles in drought-responses, including those that encode late-embryogenesis-abundant proteins thought to confer osmoprotective functions during water stress, transcription factors, heat-shock proteins as well as proteins involved in signal transduction and oxidative stress. The unigene clusters were screened for short tandem repeats for further development as microsatellite markers. A total of 592 clusters contained 646 repeats, representing 3.3% of the ESTs queried. The ESTs presented here are the first dehydration stress transcriptome of cassava and can be utilized for the development of microarrays and gene-derived molecular markers to further dissect the molecular basis of drought tolerance in cassava.
Carob (Ceratonia siliqua L.) is a trioecious evergreen legume tree native to the Middle East and widespread throughout the Mediterranean as a crop, or as part of the maquis. Amplified Fragment Length Polymorphism (AFLP) markers were used to estimate intra-specific genetic diversity among 41 carob genotypes from different areas of the eastern and western Mediterranean basin. Italian and Spanish cultivated varieties were included in the study for fingerprinting. A total of 96 unambiguous polymorphic loci were scored from seven Eco RI/Mse I primer combinations. Simple matching similarity coefficients between pairs of genotypes ranged from 0.542 to 1.000, indicating a high level of intra-specjfic diversity. AFLP markers clearly discriminated between all the accessions, except for two Sicilian varieties. An UPGMA (Unweighted Pair Group Method with Arithmetic Mean) dendrogram and a Principal Co-ordinates Analysis revealed a broad relationship between the clusters and the geographic origin of the genotypes, with few exceptions. Most of the hermaphrodite varieties belonged to the same cluster, indicating that these cultivars were probably selected in a specific area, then distributed elsewhere. Moreover, an analysis of molecular variance (AMOVA) revealed a high level of "within geographic area" differences.
The range of environmental sex determination and sex changes throughout plant taxa from bryophytes and pteridophytes to spermatophytes is reviewed. Lability in sex expression occurs in many plant taxa but only in homosporous pteridophytes is labile sex the rule. Among angiosperms, labile sex appears to be more common among dioecious and monoecious plants than among hermaphrodites. However, hermaphrodites can control allocation to male and female functions by varying the relative emphasis on pollen and ovules. A majority of plants with labile sex expression are perennials, which indicates that flexibility in sex is more important for species with long life cycles. Environmental stress, caused by less-than-optimal light, nutrition, weather or water conditions, often favours maleness. The extreme lability in the sex expression of homosporous pteridophytes is suggested to be related primarily to the mating systems.
Experiments with several Arabidopsis thaliana mutants have revealed a web of interactions between hormonal signaling. Here, we show that the Arabidopsis mutant radical-induced cell death1 (rcd1), although hypersensitive to apoplastic superoxide and ozone, is more resistant to chloroplastic superoxide formation, exhibits reduced sensitivity to abscisic acid, ethylene, and methyl jasmonate, and has altered expression of several hormonally regulated genes. Furthermore, rcd1 has higher stomatal conductance than the wild type. The rcd1-1 mutation was mapped to the gene At1g32230 where it disrupts an intron splice site resulting in a truncated protein. RCD1 belongs to the (ADP-ribosyl)transferase domain–containing subfamily of the WWE protein–protein interaction domain protein family. The results suggest that RCD1 could act as an integrative node in hormonal signaling and in the regulation of several stress-responsive genes.
Mulberry (Morus alba L.) is a dioecious plant. In vitro sex modification in mulberry by using two chemicals, viz., ethrel and silver nitrate, is described. 2-Chloroethylphosphonic acid (ethrel) and silver nitrate were filter sterilized and added to Murashige and Skoog (1962) medium supplemented with 5 M 6-benzyl amino purine (BA). Ethrel applied at different concentrations produced female, male and mixed inflorescences in male mulberry nodal cuttings. Maximum 13.6% female inflorescences were observed on media containing 2000 g l–1 ethrel. The induction of male, female and mixed inflorescences was observed in female plants after applying silver nitrate to the culture medium. The number of male inflorescences increased with the silver nitrate concentration to a maximum of 22.4% induction on 2500 g l–1. Bisexual flowers were also observed along with male and female flowers in silver nitrate treated plants.
Members of the Cucurbitaceae family display a range of sexual phenotypes including various combinations of male, female, or
bisexual flowers. Ethylene appears to be a key hormone regulating the sex determination process. Application of ethylene,
or inhibition of ethylene action, increases or decreases the number of pistil-bearing buds, respectively. Elevated levels
of ethylene production and expression of genes for ethylene biosynthesis, have been correlated with pistillate flower production.
In this study, we sought to determine the effect of modified endogenous ethylene production on sex expression by constitutively
expressing ACS (1-aminocyclopropane-1-carboxylate synthase), the first committed enzyme for ethylene biosynthesis, in transgenic melons
(Cucumis melo L.). Most melon genotypes are andromonoecious, where an initial phase of male flowers is followed by a mixture of bisexual
and male flowers. ACS melon plants showed increased ethylene production by leaves and flower buds, and increased femaleness as measured by earlier
and increased number of bisexual buds. ACS melons also had earlier and increased number of bisexual buds that matured to anthesis, suggesting that ethylene is important
not only for sex determination, but also for development of the bisexual bud to maturity. Field studies showed that ACS melons had earlier mature bisexual flowers, earlier fruit set, and increased number of fruit set on closely spaced nodes
on the main stem. These results provide a direct demonstration of the importance of endogenous ethylene production for female
reproductive processes in melon.
Ethylene has been implicated as a sex-determining hormone in cucumber: its exogenous application increases femaleness, and gynoecious genotypes were reported to produce more ethylene. In this study, three full-length ACC oxidase cDNAs were isolated from cucumber floral buds. RFLP analysis of a population that segregates for the F(femaleness) locus indicated that CS-ACO2 is linked to F at a distance of 8.7 cM. Expression of two of the genes, CS-ACO2 and CS-ACO3, was monitored in flowers, shoot tips and leaves of different sex genotypes. In situ mRNA hybridization indicated different patternsof tissue- and stage-specific expression of CS-ACO2 and CS-ACO3 in developing flowers. CS-ACO3 expression in mid-stage female flowers was localized to the nectaries, pistil and in the arrested staminoids, whereas CS-ACO2 transcript levels accumulated later and were found in placental tissue, ovary and staminoids. In male flowers, petals and nectaries expressed both genes, whereas ACO2 expression was strong in pollen of mature flowers. In young buds, strong expression was observed along developing vascular bundles. Four sex genotypes were compared for CS-ACO2 and CS-ACO3 expression in the shoot apex and young leaf. FF genotypes had higher transcript levels in leaves but lower levels in the shoot apex and in young buds, as compared to ff genotypes; the shoot-tip pattern is, therefore, inversely correlated with femaleness, and the possibility of a feedback inhibition mechanism underlying such correlation is discussed. The two CS-ACO genes studied displayed a differential response to ethrel treatment in different organs and sex genotypes, further demonstrating the complexity of the mechanisms controlling ethylene production during cucumber floral development.
A cDNA clone encoding a putative EIN3‐like protein (DC‐EIL1) was obtained from total RNA isolated from senescing carnation
(Dianthus caryophyllus L.) petals using RT‐PCR and RACE techniques. The cDNA (2382 bp) contained an open reading frame of 1986 bp corresponding
to 662 amino acids. The amino acid sequence of the N‐terminal half of the protein, ranging from 80–300 amino acid residues,
had 84% identity with that of the corresponding regions of Arabidopsis EIN3 and tobacco TEIL, although the overall identity was 49% and 52%, respectively. Northern blot analysis revealed that
the amount of mRNA corresponding to DC‐EIL1 decreased in flower tissues, especially in petals, during natural senescence and senescence induced by exogenously applied
ethylene or ABA.
In many ways, plants offer unique systems through which to study sex determination. Because the production of unisexual flowers has evolved independently in many plant species, different and novel mechanisms may be operational. Hence, there is probably not one unifying mechanism that explains sex determination in plants. Advances in our understanding of sex determination will come from the analysis of the genetics, molecular biology, and biochemistry of genes controlling sexual determination in plants. Several excellent model systems for bisexual floral development (Arabidopsis and Antirrhinum), monoecy (maize), and dioecy (Silene, asparagus, and mercury) are available for such analyses. The important questions that remain concern the mechanism of action of sex determination genes and their interrelationship, if any, with homeotic genes that determine the sexual identity of floral organ primordia. At the physiological level, the connection between hormone signaling and sexuality is not well understood, although significant correlations have been discovered. Finally, once the genes that regulate these processes are identified, cloned, and studied, new strategies for the manipulation of sexuality in plants should be forthcoming.
Microsatellite enrichment is an excess of repetitive sequences characteristic to all studied eukaryotes. It is thought to result from the accumulated effects of replication slippage mutations. Enrichment is commonly measured as the ratio of the observed frequency of microsatellites to the frequency expected to result from random association of nucleotides. We have compared enrichment of specific types of microsatellites in coding sequences with those in noncoding sequences across seven eukaryotic clades. The results reveal consistent differences between coding and noncoding regions, in terms of both the quantity of repetitive DNA and the types present. In noncoding regions, all types of microsatellite (mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats) are found in excess, and in all cases, these excesses scale in a similar exponential fashion with the length of the microsatellite. This suggests that all types of noncoding repeats are subject to similar mutational and selective processes. Coding repeats, however, appear to be under much stronger and more specific constraints. Tri- and hexanucleotide repeats are found in consistent and significant excess over a wide range of lengths in both coding and noncoding sequences, but other repeat types are much less frequent in coding regions than in noncoding regions. These findings suggest that the differences between coding and noncoding microsatellite frequencies arise from specific selection against frameshift mutations in coding regions resulting from length changes in nontriplet repeats. Furthermore, the excesses of tri- and hexanucleotide coding repeats appear to be controlled primarily by mutation pressure.
Genes for the enzymes that make plant cell wall hemicellulosic polysaccharides remain to be identified. We report here the isolation of a complementary DNA (cDNA) clone encoding one such enzyme, mannan synthase (ManS), that makes the beta-1, 4-mannan backbone of galactomannan, a hemicellulosic storage polysaccharide in guar seed endosperm walls. The soybean somatic embryos expressing ManS cDNA contained high levels of ManS activities that localized to Golgi. Phylogenetically, ManS is closest to group A of the cellulose synthase-like (Csl) sequences from Arabidopsis and rice. Our results provide the biochemical proof for the involvement of the Csl genes in beta-glycan formation in plants.
Recently, increasingly more microsatellites, or simple sequence repeats (SSRs) have been found and characterized within protein-coding genes and their untranslated regions (UTRs). These data provide useful information to study possible SSR functions. Here, we review SSR distributions within expressed sequence tags (ESTs) and genes including protein-coding, 3'-UTRs and 5'-UTRs, and introns; and discuss the consequences of SSR repeat-number changes in those regions of both prokaryotes and eukaryotes. Strong evidence shows that SSRs are nonrandomly distributed across protein-coding regions, UTRs, and introns. Substantial data indicates that SSR expansions and/or contractions in protein-coding regions can lead to a gain or loss of gene function via frameshift mutation or expanded toxic mRNA. SSR variations in 5'-UTRs could regulate gene expression by affecting transcription and translation. The SSR expansions in the 3'-UTRs cause transcription slippage and produce expanded mRNA, which can be accumulated as nuclear foci, and which can disrupt splicing and, possibly, disrupt other cellular function. Intronic SSRs can affect gene transcription, mRNA splicing, or export to cytoplasm. Triplet SSRs located in the UTRs or intron can also induce heterochromatin-mediated-like gene silencing. All these effects caused by SSR expansions or contractions within genes can eventually lead to phenotypic changes. SSRs within genes evolve through mutational processes similar to those for SSRs located in other genomic regions including replication slippage, point mutation, and recombination. These mutational processes generate DNA changes that should be connected by DNA mismatch repair (MMR) system. Mutation that has escaped from the MMR system correction would become new alleles at the SSR loci, and then regulate and/or change gene products, and eventually lead to phenotype changes. Therefore, SSRs within genes should be subjected to stronger selective pressure than other genomic regions because of their functional importance. These SSRs may provide a molecular basis for fast adaptation to environmental changes in both prokaryotes and eukaryotes.
Experiments with several Arabidopsis thaliana mutants have revealed a web of interactions between hormonal signaling. Here, we show that the Arabidopsis mutant radical-induced cell death1 (rcd1), although hypersensitive to apoplastic superoxide and ozone, is more resistant to chloroplastic superoxide formation, exhibits reduced sensitivity to abscisic acid, ethylene, and methyl jasmonate, and has altered expression of several hormonally regulated genes. Furthermore, rcd1 has higher stomatal conductance than the wild type. The rcd1-1 mutation was mapped to the gene At1g32230 where it disrupts an intron splice site resulting in a truncated protein. RCD1 belongs to the (ADP-ribosyl)transferase domain-containing subfamily of the WWE protein-protein interaction domain protein family. The results suggest that RCD1 could act as an integrative node in hormonal signaling and in the regulation of several stress-responsive genes.
Salinization poses an increasingly serious problem in coastal and agricultural areas with negative effects on plant productivity and yield. Avicennia marina is a pantropical mangrove species that can survive in highly saline conditions. As a first step towards the characterization of genes that contribute to combating salinity stress, the construction of a cDNA library of A. marina genes is reported here. Random expressed sequence tag (EST) sequencing of 1,841 clones produced 1,602 quality reads. These clones were classified into functional categories, and BLAST: comparisons revealed that 113 clones were homologous to genes earlier implicated in stress responses, of which the dehydrins are the most predominant in this category. Of the ESTs analyzed, 30% showed homology to previously uncharacterized genes in the public plant databases. Of these 30%, 52 clones were selected for reverse Northern analysis: 26 were shown to be up-regulated and five shown to be down-regulated. The results obtained by reverse Northern analysis were confirmed by Northern analysis for three clones.
Expressed sequence tag (EST) projects have generated a vast amount of publicly available sequence data from plant species; these data can be mined for simple sequence repeats (SSRs). These SSRs are useful as molecular markers because their development is inexpensive, they represent transcribed genes and a putative function can often be deduced by a homology search. Because they are derived from transcripts, they are useful for assaying the functional diversity in natural populations or germplasm collections. These markers are valuable because of their higher level of transferability to related species, and they can often be used as anchor markers for comparative mapping and evolutionary studies. They have been developed and mapped in several crop species and could prove useful for marker-assisted selection, especially when the markers reside in the genes responsible for a phenotypic trait. Applications and potential uses of EST-SSRs in plant genetics and breeding are discussed.
The phytohormone ethylene is a key regulator of plant growth and development. Components of the pathway for ethylene signal transduction were identified by genetic approaches in Arabidopsis and have now been shown to function in agronomically important plants as well.
This review focuses on recent advances in our knowledge on ethylene signal transduction, in particular on recently proposed components of the pathway, on the interaction between the pathway components and on the roles of transcriptional and post-transcriptional regulation in ethylene signalling.
Data indicate that the site of ethylene perception is at the endoplasmic reticulum and point to the importance of protein complexes in mediating the initial steps in ethylene signal transduction. The expression level of pathway components is regulated by both transcriptional and post-transcriptional mechanisms, degradation of the transcription factor EIN3 being a primary means by which the sensitivity of plants to ethylene is regulated. EIN3 also represents a control point for cross-talk with other signalling pathways, as exemplified by the effects of glucose upon its expression level. Amplification of the initial ethylene signal is likely to play a significant role in signal transduction and several mechanisms exist by which this may occur based on properties of known pathway components. Signal output from the pathway is mediated in part by carefully orchestrated changes in gene expression, the breadth of these changes now becoming clear through expression analysis using microarrays.
Using a combination of approaches, three EIN3-like (EIL) genes DC-EIL1/2 (AY728191), DC-EIL3 (AY728192), and DC-EIL4 (AY728193) were isolated from carnation (Dianthus caryophyllus) petals. DC-EIL1/2 deduced amino acid sequence shares 98% identity with the previously cloned and characterized carnation
DC-EIL1 (AF261654), 62% identity with DC-EIL3, and 60% identity with DC-EIL4. DC-EIL3 deduced amino acid sequence shares 100%
identity with a previously cloned carnation gene fragment, Dc106 (CF259543), 61% identity with Dianthus caryophyllus DC-EIL1 (AF261654), and 59% identity with DC-EIL4. DC-EIL4 shared 60% identity with DC-EIL1 (AF261654). Expression analyses
performed on vegetative and flower tissues (petals, ovaries, and styles) during growth and development and senescence (natural
and ethylene-induced) indicated that the mRNA accumulation of the DC-EIL family of genes in carnation is regulated developmentally and by ethylene. DC-EIL3 mRNA showed significant accumulation upon ethylene exposure, during flower development, and upon pollination in petals and
styles. Interestingly, decreasing levels of DC-EIL3 mRNA were found in wounded leaves and ovaries of senescing flowers whenever ethylene levels increased. Flowers treated with
sucrose showed a 2 d delay in the accumulation of DC-EIL3 transcripts when compared with control flowers. These observations suggest an important role for DC-EIL3 during growth and
development. Changes in DC-EIL1/2 and DC-EIL4 mRNA levels during flower development, and upon ethylene exposure and pollination were very similar. mRNA levels of the DC-EILs in styles of pollinated flowers showed a positive correlation with ethylene production after pollination. The cloning and
characterization of the EIN3-like genes in the present study showed their transcriptional regulation not previously observed
for EILs.
An afternoon stroll through an English garden reveals the breathtaking beauty and enormous diversity of flowering plants. The extreme variation of flower morphologies, combined with the relative simplicity of floral structures and the wealth of floral mutants available, has made the flower an excellent model for studying developmental cell-fate specification, morphogenesis and tissue patterning. Recent molecular genetic studies have begun to reveal the transcriptional regulatory cascades that control early patterning events during flower formation, the dynamics of the gene-regulatory interactions, and the complex combinatorial mechanisms that create a distinct final floral architecture and form.
The endemic Hawaiian mints represent a major island radiation that likely originated from hybridization between two North American polyploid lineages. In contrast with the extensive morphological and ecological diversity among taxa, ribosomal DNA sequence variation has been found to be remarkably low. In the past few years, expressed sequence tag (EST) projects on plant species have generated a vast amount of publicly available sequence data that can be mined for simple sequence repeats (SSRs). However, these EST projects have largely focused on crop or otherwise economically important plants, and so far only few studies have been published on the use of intragenic SSRs in natural plant populations. We constructed an EST library from developing fleshy nutlets of Stenogyne rugosa principally to identify genetic markers for the Hawaiian endemic mints.
The Stenogyne fruit EST library consisted of 628 unique transcripts derived from 942 high quality ESTs, with 68% of unigenes matching Arabidopsis genes. Relative frequencies of Gene Ontology functional categories were broadly representative of the Arabidopsis proteome. Many unigenes were identified as putative homologs of genes that are active during plant reproductive development. A comparison between unigenes from Stenogyne and tomato (both asterid angiosperms) revealed many homologs that may be relevant for fruit development. Among the 628 unigenes, a total of 44 potentially useful microsatellite loci were predicted. Several of these were successfully tested for cross-transferability to other Hawaiian mint species, and at least five of these demonstrated interesting patterns of polymorphism across a large sample of Hawaiian mints as well as close North American relatives in the genus Stachys.
Analysis of this relatively small EST library illustrated a broad GO functional representation. Many unigenes could be annotated to involvement in reproductive development. Furthermore, first tests of microsatellite primer pairs have proven promising for the use of Stenogyne rugosa EST SSRs for evolutionary and phylogeographic studies of the Hawaiian endemic mints and their close relatives. Given that allelic repeat length variation in developmental genes of other organisms has been linked with morphological evolution, these SSRs may also prove useful for analyses of phenotypic differences among Hawaiian mints.
Due to its origin, peanut has a very narrow genetic background. Wild relatives can be a source of genetic variability for cultivated peanut. In this study, the transcriptome of the wild species Arachis stenosperma accession V10309 was analyzed.
ESTs were produced from four cDNA libraries of RNAs extracted from leaves and roots of A. stenosperma. Randomly selected cDNA clones were sequenced to generate 8,785 ESTs, of which 6,264 (71.3%) had high quality, with 3,500 clusters: 963 contigs and 2537 singlets. Only 55.9% matched homologous sequences of known genes. ESTs were classified into 23 different categories according to putative protein functions. Numerous sequences related to disease resistance, drought tolerance and human health were identified. Two hundred and six microsatellites were found and markers have been developed for 188 of these. The microsatellite profile was analyzed and compared to other transcribed and genomic sequence data.
This is, to date, the first report on the analysis of transcriptome of a wild relative of peanut. The ESTs produced in this study are a valuable resource for gene discovery, the characterization of new wild alleles, and for marker development. The ESTs were released in the [GenBank:EH041934 to EH048197].
Cassava (Manihot esculenta Crantz) is a staple food for over 600 million people in the tropics and subtropics and is increasingly used as an industrial crop for starch production. Cassava has a high growth rate under optimal conditions but also performs well in drought-prone areas and on marginal soils. To increase the tools for understanding and manipulating drought tolerance in cassava, we generated expressed sequence tags (ESTs) from normalized cDNA libraries prepared from dehydration-stressed and control well-watered tissues. Analysis of a total of 18,166 ESTs resulted in the identification of 8,577 unique gene clusters (5,383 singletons and 3,194 clusters). Functional categories could be assigned to 63% of the unigenes, while another approximately 11% were homologous to hypothetical genes with unclear functions. The remaining approximately 26% were not significantly homologous to sequences in public databases suggesting that some may be novel and putatively specific to cassava. The dehydration-stressed library uncovered numerous ESTs with recognized roles in drought-responses, including those that encode late-embryogenesis-abundant proteins thought to confer osmoprotective functions during water stress, transcription factors, heat-shock proteins as well as proteins involved in signal transduction and oxidative stress. The unigene clusters were screened for short tandem repeats for further development as microsatellite markers. A total of 592 clusters contained 646 repeats, representing 3.3% of the ESTs queried. The ESTs presented here are the first dehydration stress transcriptome of cassava and can be utilized for the development of microarrays and gene-derived molecular markers to further dissect the molecular basis of drought tolerance in cassava.
Designing PCR and sequencing primers are essential activities for molecular biologists around the world. This chapter assumes acquaintance with the principles and practice of PCR, as outlined in, for example, refs. 1, 2, 3, 4.
The flowers of Ceratonia siliqua, an anomalous caesalpinioid legume in the tribe Cassieae, are unusual in being unisexual and in lacking petals. Inflorescence development, organogeny, and flower development are described for this species. All flowers are originally bisexual, but one sex is suppressed during late development of functionally male and female flowers. Ceratonia siliqua is highly plastic in sexuality of individuals, inflorescence branching pattern, racemose or cymose inflorescences, bracteole presence, terminal flower presence, organ number per whorl, missing floral organs, pollen grain form, and carpel cleft orientation. Order of initiation is: five sepals in helical order, then five stamens in helical order together with the carpel. Each stamen is initiated as two alternisepalous primordia that fuse to become a continuous antesepalous ridge; in some flowers, the last one or two stamens of the five may form as individual antesepalous mounds. Petal rudiments are occasional in mature flowers. Position of organs is atypical: the median sepal is on the adaxial side in Ceratonia, rather than abaxial as in most other caesalpinioids. This feature in Ceratonia may be viewed as a link to subfamily Mimosoideae, in which this character state is constant.
The application of Ethrel (2-chloroethane phosphonic acid), an ethylene-releasing compound, to monoecious cultivars of cucumber and squash and an andromonoecious cultivar of muskmelon, caused a shift towards femaleness in all three species. The increase in femaleness manifested itself in several symptoms: a decrease in the number of staminate (male) flowers, an increase in the number of pistillate (female) or hermaphrodite (perfect) flowers, and a change in flowering pattern, namely, formation of female flowers at lower nodes in cucumber and squash, and formation of hermaphrodite flowers on the main axis in muskmelon where normally only male flowers are formed in this cultivar.
Apical application of silver nitrate (AgNO3; 50 and 100 μg per plant) and silver thiosulphate anionic complex (Ag(S2O3) 2 (3-) ; STS; 25, 50 and 100 μg per plant) to female plants of Cannabis sativa induced the formation of reduced male, intersexual and fully altered male flowers on the newly formed primary lateral branches (PLBs); 10 μg per plant of AgNO3 was ineffective and 150 μg treatment proved inhibitory. A maximum number of fully altered male flowers were formed in response to 100 μg STS. The induced male flowers produced pollen grains that germinated on stigmas and effected seed set. Silver ion applied as STS was more effective than AgNO3 in inducing flowers of altered sex. The induction of male flowers on female plants demonstrated in this work is useful for producing seeds that give rise to only female plants. This technique is also useful for maintaining gynoecious lines.
Although most flowering plant species are hermaphrodites, there are still a large number of species with unisexual flowers. Classical geneticists identified the genes that distinguish the sexes in many species, but, until recently, nothing was known about the molecular mechanisms involved. Knowledge of the genes that control floral organ identity in a broad range of angiosperms has now allowed detailed comparative expression studies. Intriguingly, the sex-related differences indicate that the mechanism of sex determination is not the same in all species. In addition, genes that affect the sexual expression of flowers have been cloned from maize, and the sequences of these indicate that they are involved in hormone metabolism.
The flowers of Ceratonia siliqua, an anomalous caesalpinioid legume in the tribe Cassieae, are unusual in being unisexual and in lacking petals. Inflorescence development, organogeny, and flower development are described for this species. All flowers are originally bisexual, but one sex is suppressed during late development of functionally male and female flowers. Ceratonia siliqua is highly plastic in sexuality of individuals, inflorescence branching pattern, racemose or cymose inflorescences, bracteole presence, terminal flower presence, organ number per whorl, missing floral organs, pollen grain form, and carpel cleft orientation. Order of initiation is: five sepals in helical order, then five stamens in helical order together with the carpel. Each stamen is initiated as two alternisepalous primordia that fuse to become a continuous antesepalous ridge; in some flowers, the last one or two stamens of the five may form as individual antesepalous mounds. Petal rudiments are occasional in mature flowers. Position of organs is atypical: the median sepal is on the adaxial side in Ceratonia, rather than abaxial as in most other caesalpinioids. This feature in Ceratonia may be viewed as a link to subfamily Mimosoideae, in which this character state is constant.
The variability of isozymes in nine enzyme systems was studied in 25 carob (Ceratonia siliqua L.) cultivars using starch gel electrophoresis of leaf extracts. Five enzymes (phosphoglucomutase, phosphoglucoisomerase, aspartate aminotransferase, shikimic dehydrogenase, and aconitase) were polymorphic, making it possible for the 25 cultivars to be classified into eight phenotype categories.
New molecular markers derived from expressed sequence tag (EST) sequences were mapped on linkage maps of Italian ryegrass by a two-way pseudo-testcross strategy. cDNA sequences were obtained from various tissues of Italian ryegrass (Lolium multiflorum) and converted into cleaved amplified polymorphic sequence (CAPS) markers. Of 260 EST primer pairs that amplified a single band, 74 generated bands that showed clear polymorphisms among individuals of an F1 mapping family. Of the 74 polymorphic marker loci, 69 were mapped on an Italian ryegrass linkage map previously constructed using amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), and simple sequence repeat (SSR) markers. The newly-developed EST-CAPS markers would be useful as an efficient tool to identify genetic markers and to identify candidate genes for quantitative trait loci (QTLs) associated with important traits in Italian ryegrass.
Ethylene production, level of 1-aminocyclopropane-1-carboxylic acid (ACC) and activity of the ethylene forming enzyme (EFE) were higher in apices of gynoecious cucumber (Cucumis sativus cv. Alma) as compared to monoecious cucumber (C. sativus cv. Elem). Application of indole-3-acetic acid (IAA) enhanced ethylene and ACC production in both cultivars. The stimulatory effect of IAA was more pronounced in gynoecious apices. Induction of ethylene production and accumulation of ACC resulting from treatment with IAA were effectively blocked by aminoethoxyvinylglycine (AVG). Content of endogenous IAA, measured by an enzyme immunoassay, was lower in gynoecious cucumber as compared to monoecious one. Treatment of gynoecious plants with the antiauxins α-(p-chlorophenoxy)isobutyric acid (PCIB) and β-naphthaleneacetic acid (β-NAA) did not inhibit female sex expression.
It appears that although exogenous IAA enhances ACC and ethylene production, endogenous IAA might not have a major role in the control of sex expression in cucumber of the Beit-Alfa type.
A review is presented of the recent developments in the metabolism andfunction of polyamines in plants. Polyamines appear to be involved in a widerange of plant processes so their exact role is not completely understood. Inthis review, the metabolic pathways involved in polyamine biosynthesis anddegradation are explained, along with the transport and conjugation of thesecompounds. The methodologies involved in the analysis of polyamine functionusing metabolic inhibitors and genetic and molecular approaches are described.The occurrence and distribution of polyamine-derived alkaloids are also dealtwith. The direction of future research in the study of plant polyamines isindicated.
The biosynthesis of the gaseous phytohormone ethylene is a highly regulated process. A major point of regulation occurs at
the generally rate-limiting step in biosynthesis, catalyzed by the enzyme ACC synthase (ACS). ACS is encoded by a multigene
family, and different members show distinct patterns of expression during growth and development, and in response to various
external cues. In addition to this transcriptional control, the stability of the ACS protein is also highly regulated. Here
we review these two distinct regulatory inputs that control the spatial and temporal patterns of ethylene biosynthesis.
Simple sequence repeat (SSR) markers generated from expressed sequence tag (EST) sequences represent useful tools for genotyping
and their development is relatively easy because of the public availability of EST databases. We report design and application
of EST–SSRs to assess the level of genetic diversity among thirty-five asparagus cultivars and to fingerprint DePaoli, a new
variety released by University of California, Riverside. DNA was isolated from bulks of pooled cladophylls coming from five
plants of each variety to reduce the number of DNA extractions and PCR reactions. Allele frequencies were estimated from the
intensity of the bands in two bulks and two individual plant samples for each variety. Although asparagus varieties derive
from a limited germplasm pool, eight EST–SSR loci differentiated all of the analyzed cultivars. Moreover, UPGMA (unweighted
pair group method with arithmetic mean) and neighbor-joining trees, as well as principal components analysis separated the
cultivars into clusters corresponding to the geographical areas where they originated.
The last two centuries witnessed the human-caused fragmentation of Tunisian Ceratonia siliqua L. (Caesalpinoideae) populations which were often represented by scattered individuals. Seventeen populations growing in four bioclimatic zones: sub-humid, upper semi-arid, mean semi-arid and lower semi-arid zones, were sampled for allozyme diversity to assess their genetic diversity and structuration using eight isozymes revealed by starch gel electrophoresis. The species showed high diversity within populations. The average number of alleles per polymorphic locus was 1.98, the percentage of polymorphic loci was 83.4% and the mean observed heterozygosity (Ho) and expected heterozygosity (He) under Hardy–Weinberg equilibrium were respectively 0.247 and 0.316. A substantial level of inbreeding within populations induced by Wahlund effect, was observed (F
IS = 0.231). High diversity resulted from the great number of genotypes in the ancestral population before fragmentation, favoured by the outbreeding of the species. High differentiation and low gene flow were detected among populations (F
ST = 0.200) and among pairs of ecological zones (0.113< F
ST <0.198). However, the differentiation coefficient of the four zones was low (F
ST = 0.085) and similar to the average F
ST for forest trees. Population structuration depends on geographic distance between sites rather than bioclimate, indicating that structuration has occurred slowly and that climatic conditions have had little effect. Nei's genetic distances (D) between populations were low and ranged from 0.004 to 0.201. Mean D value for all population was 0.087. The UPGMA clustering established for all populations through Nei's genetic distances did not clearly show that, for the majority of populations, grouping had resulted from ecological factors or geographic location. The substantial differentiation and the high genetic similarities between populations indicate that populations have been recently isolated as a result of anthropic pressure. In-situ conservation strategies must first focus on populations with a high level of genetic diversity and rare alleles. Appropriate conservation action should take account of bioclimatic zones. Ex-situ preservation should be based on a maximum number of individuals collected within populations in each ecological group and their propagation in different bioclimates by means of cuttings.
Ethyl hydrogen-1-propylphosphonate (Niagara 10637) at concentrations of 500, 1000 and 2000 mg/l had two different effects on sex expression ofRicinus communis L. cvIari 132. Higher concentrations (1000 and 2000 mg/l), applied just before emergence of inflorescence i.e. to 90 day old plants, induced a marked increase in female flower number
and reduced the number of male flowers on the first inflorescence. However, there was a tendency towards maleness on the second
and third inflorescences. When applied to 60 day old plants, the test compound induced a high degree of maleness on the first
and second inflorescences. Sex expression was not markedly altered with treatments at still younger stages (seeds and seedlings).
The majority of the world's flowering plants are hermaphrodite but many of them encourage cross pollination by means of spatial or temporal separation of eggs and pollen, or by genetically-controlled physiological incompatibility. A minority of species has taken the avoidance of self-pollination to its logical conclusion by evolving two distinct and sexually different forms (dioecy). In a very small number of plants, dioecy has been accompanied by the development of sex chromosomes. From the study of the development of male and female flowers of different species it is clear that there is no common underlying mechanism and that sex determination systems leading to dioecy have originated independently many times in evolution. This Botanical Briefing highlights new information from recent molecular approaches in the study of dioecy. Copyright 2000 Annals of Botany Company
Ethrel treatment at 960, 1920, and 3840 parts 10−6 to male plants of M. nigra produced intersex and female flowers. The intersexual flowers showed various degrees of transformation of male sex organs
into female. The complete female flowers produced were similar to female controls. Fruit setting occurred in inflorescences
having both types of flowers which were similar in appearance but smaller in size than the untreated control.
The use of short tandem repeat polymorphisms (STRPs) as marker loci for linkage analysis is becoming increasingly important due to their large numbers in the human genome and their high degree of polymorphism. Fluorescence-based detection of the STRP pattern with an automated DNA sequencer has improved the efficiency of this technique by eliminating the need for radioactivity and producing a digitized autoradiogram-like image that can be used for computer analysis. In an effort to simplify the procedure and to reduce the cost of fluorescence STRP analysis, we have developed a technique known as multiplexing STRPs with tailed primers (MSTP) using primers that have a 19-bp extension, identical to the sequence of an M13 sequencing primer, on the 5' end of the forward primer in conjunction with multiplexing several primer pairs in a single polymerase chain reaction (PCR) amplification. The banding pattern is detected with the addition of the M13 primer-dye conjugate as the sole primer conjugated to the fluorescent dye, eliminating the need for direct conjugation of the infrared fluorescent dye to the STRP primers. The use of MSTP for linkage analysis greatly reduces the number of PCR reactions. Up to five primer pairs can be multiplexed together in the same reaction. At present, a set of 148 STRP markers spaced at an average genetic distance of 28 cM throughout the autosomal genome can be analyzed in 37 sets of multiplexed amplification reactions. We have automated the analysis of these patterns for linkage using software that both detects the STRP banding pattern and determines their sizes. This information can then be exported in a user-defined format from a database manager for linkage analysis.
Ethylene regulates a multitude of plant processes, ranging from seed germination to organ senescence. Of particular economic importance is the role of ethylene as an inducer of fruit ripening. Ethylene is synthesized from S-adenosyl-L-methionine via 1-aminocyclopropane-1-carboxylic acid (ACC). The enzymes catalyzing the two reactions in this pathway are ACC synthase and ACC oxidase. Environmental and endogenous signals regulate ethylene biosynthesis primarily through differential expression of ACC synthase genes. Components of the ethylene signal transduction pathway have been identified by characterization of ethylene-response mutants in Arabidopsis thaliana. One class of mutations, exemplified by etr1, led to the identification of the ethylene receptors, which turned out to be related to bacterial two-component signaling systems. Mutations that eliminate ethylene binding to the receptor yield a dominant, ethylene-insensitive phenotype. CTR1 encodes a Raf-like Ser/Thr protein kinase that acts downstream from the ethylene receptor and may be part of a MAP kinase cascade. Mutants in CTR1 exhibit a constitutive ethylene-response phenotype. Both the ethylene receptors and CTR1 are negative regulators of ethylene responses. EIN2 and EIN3 are epistatic to CTR1, and mutations in either gene lead to ethylene insensitivity. Whereas the function of EIN2 in ethylene transduction is not known, EIN3 is a putative transcription factor involved in regulating expression of ethylene-responsive genes. Biotechnological modifications of ethylene synthesis and of sensitivity to ethylene are promising methods to prevent spoilage of agricultural products such as fruits, whose ripening is induced by ethylene.
Despite its simple two-carbon structure, the olefin ethylene is a potent modulator of plant growth and development (Ecker, 1995). The plant hormone ethylene is involved in many aspects of the plant life cycle, including seed germination, root hair development, root nodulation, flower senescence, abscission, and fruit ripening (reviewed in Johnson and Ecker, 1998). The production of ethylene is tightly regulated by internal signals during development and in response to environmental stimuli from biotic (e.g., pathogen attack) and abiotic stresses, such as wounding, hypoxia, ozone, chilling, or freezing. To understand the roles of ethylene in plant functions, it is important to know how this gaseous hormone is synthesized, how its production is regulated, and how the signal is transduced. Morphological changes in dark-grown (etiolated) seedlings treated with ethylene or its metabolic precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), have been termed the triple response. The exaggerated curvature of the apical hook, radial swelling of the hypocotyl, and shortening of the hypocotyl and root are the unmistakable hallmarks of this ethylene response. Over the past decade, the triple response phenotype has been used to screen for mutants that are defective in ethylene responses (Bleecker et al., 1988; Guzman and Ecker, 1990). Etiolated Arabidopsis seedlings with minor or no phenotypic response upon ethylene application are termed ethylene-insensitive (ein) or ethylene-resistant (etr) mutants. Mutants have also been identified that display a constitutive triple response in the absence of ethylene (Kieber et al., 1993; Roman and Ecker, 1995). This class can be divided into subgroups based on whether or not the constitutive triple response can be suppressed by inhibitors of ethylene perception and biosynthesis, such as silver thiosulfate and aminoethoxyvinyl glycine (AVG). Mutants that are unaffected by these inhibitors are termed constitutive triple-response (ctr) mutants, whereas mutants whose phenotype reverts to normal morphology are termed ethylene-overproducer (eto) mutants, which are defective in the regulation of hormone biosynthesis. The genetic hierarchy among ethylene biosynthesis and signaling pathway components in Arabidopsis has been established by epistasis analysis using these mutants (Solano and Ecker, 1998; Stepanova and Ecker, 2000).
The intent of this review is not to cover all aspects of ethylene biology but to focus on recent findings. In particular, we examine interaction of ethylene and two other plant growth regulators, jasmonic acid (JA) and salicyclic acid (SA), and their roles in mediating responses to biotic and abiotic stresses. We begin by summarizing what is currently known about the mechanism and regulation of ethylene biosynthesis and by providing an update of our current understanding of the ethylene signaling pathway.
A growing array of sequence-based tools is helping to reveal the organization, evolution and syntenic relationships of legume genomes. The results indicate that legumes form a coherent taxonomic group with frequent and widespread macro- and microsynteny. This is good news for two model legume systems, Medicago truncatula and Lotus japonicus. Indeed, both models have recently been used to clone and characterize genes for nodulation-related receptors that were originally described in legumes with more complex genomes. Studies of legume genomes have also provided insight into genome size, gene clustering, genome duplications and repetitive elements. To understand legume genomes better, it will be necessary to develop tools for studying under-represented taxa beyond the relatively small group of economically important species that have been examined so far.
Over three million sequences from approximately 200 plant species have been deposited in the publicly available plant expressed sequence tag (EST) sequence databases. Many of the ESTs have been sequenced as an alternative to complete genome sequencing or as a substrate for cDNA array-based expression analyses. This creates a formidable resource from both biodiversity and gene-discovery standpoints. Bioinformatics-based sequence analysis tools have extended the scope of EST analysis into the fields of proteomics, marker development and genome annotation. Although EST collections are certainly no substitute for a whole genome scaffold, this "poor man's genome" resource forms the core foundations for various genome-scale experiments within the as yet unsequenceable plant genomes.
Freely available computer programs were arranged in a pipeline to extract microsatellites from public citrus EST sequences, retrieved from the NCBI. In total, 3,278 bi- to hexa-type SSR-containing sequences were identified from 56,199 citrus ESTs. On an average, one SSR was found per 5.2 kb of EST sequence, with the tri-nucleotide motifs as the most abundant. Primer sequences flanking SSR motifs were successfully identified from 2,295 citrus ESTs. Among those, a subset (100 pairs) were synthesized and tested to determine polymorphism and heterozygosity between/within two genera, sweet orange (C. sinensis) and Poncirus (P. trifoliata), which are the parents of the citrus core mapping population selected for an international citrus genomics effort. Eighty-seven pairs of primers gave PCR amplification to the anticipated SSRs, of which 52 and 35 appear to be homozygous and heterozygous, respectively, in sweet orange, and 67 and 20, respectively, in Poncirus. By pairing the loci between the two intergeneric species, it was found that 40 are heterozygous in at least one species with two alleles (9), three alleles (28), or four alleles (3), and the remaining 47 are homozygous in both species with either one allele (31) or two alleles (16). These EST-derived SSRs can be a resource used for understanding of the citrus SSR distribution and frequency, and development of citrus EST-SSR genetic and physical maps. These SSR primer sequences are available upon request.
This chapter focuses on the hormonal regulation of sex expression in monoecious plants, with particular emphasis on maize (Z. mays) and cucumber (C. sativus). It also discusses the mutual and different aspects of the regulatory systems that control their sex expression and presents a genetic model of sex expression in maize and cucumber plants. In maize plants, gibberellins act to arrest the stamen primordia in the primary and secondary florets of the ears, and low levels of gibberellins do not cause the arrest of pistil primordia in the primary florets of the ears. In principle, gibberellins do not affect the development of pistil primordial as evidenced by the analysis of dwarf (d) mutants. On the contrary, in cucumber plants, ethylene acts on both the development of pistil primordia and the arrest of stamen primordia, which results in the induction of femaleness. Therefore, ethylene has opposing effects on the development of sexual organs, stamens, and pistils. With regard to the hormonal regulation of sex expression, it is interesting that both plant hormones, gibberellins and ethylene, cause the arrest of stamen primordial in maize and cucumber, respectively. This indicates that ethylene‐ and gibberellins‐signaling pathways mediate the arrest (by PCD) of stamen primordia in maize and cucumber.
Ethylene plays a key role in sex determination of cucumber flowers. Gynoecious cucumber shoots produce more ethylene than
monoecious shoots. Because monoecious cucumbers produce both male and female flower buds in the shoot apex and because the
relative proportions of male and female flowers vary due to growing conditions, the question arises as to whether the regulation
of ethylene biosynthesis in each flower bud determines the sex of the flower. Therefore, the expression of a 1-aminocyclopropane-1-carboxylic
acid synthase gene, CS-ACS2, was examined in cucumber flower buds at different stages of development. The results revealed that CS-ACS2 mRNA began to accumulate just beneath the pistil primordia of flower buds at the bisexual stage, but was not detected prior
to the formation of the pistil primordia. In buds determined to develop as female flowers, CS-ACS2 mRNA continued to accumulate in the central region of the developing ovary where ovules and placenta form. In gynoecious
cucumber plants that produce only female flowers, accumulation of CS-ACS2 mRNA was detected in all flower buds at the bisexual stage and at later developmental stages. In monoecious cucumber, flower
buds situated on some nodes accumulated CS-ACS2 mRNA, but others did not. The proportion of male and female flowers in monoecious cucumbers varied depending on the growth
conditions, but was correlated with changes in accumulation of CS-ACS2 mRNA in flower buds. These results demonstrate that CS-ACS2-mediated biosynthesis of ethylene in individual flower buds is
associated with the differentiation and development of female flowers.
Through multifaceted genome-scale research involving phylogenomics, targeted gene surveys, and gene expression analyses in diverse basal lineages of angiosperms, our studies provide insights into the most recent common ancestor of all extant flowering plants. MADS-box gene duplications have played an important role in the origin and diversification of angiosperms. Furthermore, early angiosperms possessed a diverse tool kit of floral genes and exhibited developmental 'flexibility', with broader patterns of expression of key floral organ identity genes than are found in eudicots. In particular, homologs of B-function MADS-box genes are more broadly expressed across the floral meristem in basal lineages. These results prompted formulation of the 'fading borders' model, which states that the gradual transitions in floral organ morphology observed in some basal angiosperms (e.g. Amborella) result from a gradient in the level of expression of floral organ identity genes across the developing floral meristem.
Boys and girls come out to play: the molecular biology of dioecious plants Regulation of ethylene biosynthesis
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C (2000) Boys and girls come out to play: the molecular biology of dioecious plants. Ann Bot 86:211–221 Argueso CT, Hansen M, Kieber JJ (2007) Regulation of ethylene biosynthesis. J Plant Growth Regul 26:92–105 Batlle