Marco Fambrini

Expression of a dehydrin gene during embryo development and drought stress in ABA-deficient mutants of sunflower (Helianthus annuus L.)

The synthesis of a particular class of proteins, the dehydrins, is a common response to drought in plants. Dehydrins are known to be synthesized by the cell in response to abscisic acid, which represents a link between environment and nuclear activity, though dehydrin genes may be expressed even constitutively. We have investigated the relationship between abscisic acid (ABA) and accumulation of a dehydrin mRNA in sunflower, in which a dehydrin cDNA (HaDhn1a) was isolated. In particular, we studied changes in the steady-state level of dehydrin transcripts in two mutants for ABA synthesis and accumulation: nd-1 (an albino, non-dormant and lethal mutant with a very low ABA content and no ABA accumulation in response to stress) and w-1 (a wilty mutant, with reduced ABA accumulation) during embryo and plantlet development and drought stress. Differences between genotypes were observed through embryogenesis: w-1 shows a lower content of dehydrin transcripts in the early stages compared to control plants, indicating that ABA affects dehydrin mRNA accumulation; however, dehydrin transcripts level appears independent of ABA content in late embryogenesis. Also during drought stress in w-1 adult leaves, ABA is not quantitatively related to the steady-state level of the HaDhn1a transcripts. Finally, data on nd-1 mutant show a high level of dehydrin transcripts after drought stress in plantlet cotyledons and leaflets. These results indicate the existence of two regulation pathways of HaDhn1a transcripts accumulation, an ABA-dependent and an ABA-independent one, which may have cumulative effects.

Characterization of LEAFY COTYLEDON1-LIKE gene in Helianthus annuus and its relationship with zygotic and somatic embryogenesis

The Helianthus annuusLEAFY COTYLEDON1-LIKE (HaL1L) gene encodes a heme-activated protein 3 subunit of the CCAAT box-binding factor. The phylogenetic analysis indicates that HaL1L is closely related to LEAFY COTYLEDON1 (LEC1)-type of Arabidopsis thaliana. In particular, the peptide results homologous to the LEC1-LIKE gene of A. thaliana, with which it shares a high amino acid sequence identity (56%). HaL1L transcripts are accumulated primarily at an early stage of sunflower embryogenesis. High levels of HaL1L messenger RNA (mRNA) have been detected in the developing embryo proper, suspensor, endosperm, integument, and integumentary tapetum cells, while in unfertilized ovules, HaL1L mRNA was present at rather low levels. In an attempt to examine the involvement of HaL1L on somatic embryogenesis, a somaclonal variant of H. annuus × H. tuberosus (EMB-2) that produces ectopic embryo- and shoot-like structures, arranged in clusters along leaf veins, was used. We found that the epiphyllous proliferation of ectopic embryos on EMB-2 leaves was associated to HaL1L mRNA accumulation. The detection of HaL1L transcripts was evident in somatic embryos at the heart- and early cotyledon-stage. On the contrary, no signal related to HaL1L transcript accumulation was observed in EMB-2 leaves characterized by the presence of shoot-like structures. Together, these results support the conclusion that the transcription of the HaL1L gene is maintained both in zygotic and in somatic embryogenesis. In addition, the ectopic accumulation of HaL1L mRNA in parenchymal cells around the vascular bundles of epiphyllous leaves opens the possibility that HaL1L could also be involved in switching somatic cell fate towards embryogenic competence.

A transposon-mediate inactivation of a CYCLOIDEA-like gene originates polysymmetric and androgynous ray flowers in Helianthus annuus

In several eudicots, including members of the Asteraceae family, the CYCLOIDEA (CYC) genes, which belong to the TCP class of transcription factors, are key players for floral symmetry. The sunflower inflorescence is heterogamous (radiate capitulum) with sterile monosymmetric ray flowers located in the outermost whorl of the inflorescence and hermaphrodite polysymmetric disk flowers. In inflorescence of Heliantheae tribe, flower primordia development initiates from the marginal ray flowers while disk flowers develop later in an acropetal fashion in organized parastichies along a number found to be one of Fibonacci patterns. Mutants for inflorescence morphology can provide information on the role of CYC-like genes in radiate capitulum evolution. The tubular ray flower (turf) mutant of sunflower shows hermaphrodite ray flowers with a nearly polysymmetric tubular-like corolla. Here, we demonstrate that this mutation is caused by the insertion in the TCP motif of a sunflower CYC-like gene (HaCYC2c) of non-autonomous transposable element (TE), belonging to the CACTA superfamily of transposons. We named this element Transposable element of turf1 (Tetu1). The Tetu1 insertion changes the reading frame of turf-HaCYC2c for the encoded protein and leads to a premature stop codon. Although in Tetu1 a transposase gene is lacking, our results clearly suggest that it is an active TE. The excision of Tetu1 restores the wild type phenotype or generates stable mutants. Co-segregation and sequence analysis in progenies of F2 and self-fertilized plants derived from reversion of turf to wild type clearly identify HaCYC2c as a key regulator of ray flowers symmetry. Also, HaCYC2c loss-of-function promotes the developmental switch from sterile to hermaphrodite flowers, revealing a novel and unexpected role for a CYC-like gene in the repression of female organs.

Characterization of a sunflower (Helianthus annuus L.) mutant, deficient in carotenoid synthesis and abscisic-acid content, induced by in-vitro tissue culture.

Genetic variation induced by tissue culture has been characterized in many species. The present study was conducted to genetically and phenotypically characterize an albino mutant in sunflower induced by in-vitro culture. A single recessive gene defective in carotenoid biosynthesis eventually leads to a chlorophyll loss due to photobleaching, absence of seed dormancy, and a low level of endogenous abscisic acid (ABA) in cotyledons and leaves. Further characterization has shown that the endogenous level of the hormone does not increase after drought stress and that the mutation prevents anthocyanin synthesis.

Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus × H. tuberosus

BACKGROUND AND AIMS The clone EMB-2 of the interspecific hybrid Helianthus annuus x H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence. METHODS Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography-mass spectrometry-selected ion monitoring method and an immuno-cytochemical approach. KEY RESULTS Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency. CONCLUSIONS In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone.

Genomic alterations in the interspecific hybrid Helianthus annuus×Helianthus tuberosus

Abstract The genome of a Helianthus annuus (2n=34) ×Helianthus tuberosus (2n=102) hybrid was studied at cytological, biochemical and molecular levels and compared to those of the parental species. Cytophotometric analyses showed that the hybrid has a 4C DNA content higher than expected and with a larger variability than in the parents. This high variability is probably not related to chromosome-number variations since the hybrid always had 2n=68 chromosomes. Moreover, hybrid interphase nuclei showed lower heterochromatin condensation than the parental ones. Thermal denaturation of genomic DNAs indicated that quantitative variation of some DNA families occurred in the hybrids compared to parents. Finally, molecular analyses of DNAs restricted with different enzymes, after Southern blotting and hybridization with HR probes, showed restriction patterns in the hybrid different from those observed in parents. These results indicate that interspecific hybridization between H. annuus and H. tuberosus may determine quantitative variation of some DNA families and differential DNA methylations that probably modify the nuclear structure. These phenomena are probable responses to a “genomic shock” following the interspecific cross.

Development of somatic embryos from morphogenetic cells of the interspecific hybrid Helianthus annuus * Helianthus tuberosus

Abstract The morphogenetic competence to differentiate somatic embryos was evaluated in leaf explants of anther-derived plants (EMB-1 clone) of the tetraploid interspecific hybrid Helianthus annuus × Helianthus tuberosus. Various combinations of growth regulators were compared. Embryos rapidly formed at high frequency in each media tested, even without growth regulators. Histological analysis showed repeated divisions of cells of the upper epidermis showing densely stained cytoplasm. After 6 days of culture organized structures were observed on the leaf surface. Further, these clusters of cells increased their volume by division; embryoids developed asynchronously in large numbers over small areas of the leaf. Somatic embryos appeared variously malformed and sometimes they proliferated to produce additional accessory embryos from one morphogenetic unit. Nevertheless, phenotypically normal plants were easily recovered from somatic embryos. The direct embryogenesis observed in each media tested indicates that the somatic cells of EMB-1 plants are already predetermined as embryogenic cells. These cells do not lose their wide competence in the differentiation process and retain the full morphogenetic potential typical of the zygotic cell. This assumption is strengthened by the ability of leaf explants to differentiate embryos without signals of hormonal nature.

Somaclonal variation for resistance to Verticillium dahliae in potato (Solanum tuberosum L.) plants regenerated from callus

SummaryPlant tissue culture is recognized as an important tool to generate useful genetic variability for crop improvement. Regenerated plants from callus induced from stem explants of Solanum tuberosum cv Désirée were assessed by in vitro selection, for resistance to Verticillium dahliae. This fungus is the causal agent of Verticillium wilt, a serious vascular wilt disease both in crops and wild species.The rate of in vitro multiplication by single node cuttings was used as a parameter of screening in two selection cycles with different concentrations of V. dahliae filtrate. One resistant clone was selected and then evaluated by inoculation in the growth chamber. Induced damage, and morphological traits (dry weight, leaf area and tuber production) were estimated. The selected clone was comparable to the resistant control, cv Kondor.The results suggest that genetic variation induced in tissue culture cound be utilized to generate disease resistance.

Molecular Cloning and Organ-Specific Expression of Two Gibberellin 20-Oxidase Genes of Helianthus annuus

The activity of GA 20-oxidase (GA20ox), a 2-oxoglutarate-dependent dioxygenase, is a critical regulatory factor in the gibberellin-(GA)-biosynthetic pathway. Two genes, HaGA20ox1 and HaGA20ox2, along with their corresponding cDNAs exhibiting structural features and homology to GA 20-oxidases of several plant species, have been isolated from vegetative shoots of sunflower (Helianthus annuus). Sequence analysis revealed that both genes consist of three exons and two introns. GA20ox genes from dicot and monocot species showed similar structures. The amino acid sequences deduced from the sunflower cDNA clones showed high level of similarity, with identities of HaGA20ox1/HaGA20ox2 of 84%. The phylogenetic analysis indicated that all monocot and all dicot GA20ox were grouped in two separate clusters. Within the dicot clade, HaGA20ox1 and HaGA20ox2 formed a subclade with GA 20-oxidases from Lactuca sativa (Ls20ox1 and Ls20ox2), Chrysanthemum x morifolium (DgGA20ox1), and Daucus carota (DcGA20ox2). Both HaGA20ox1 and HaGA20ox2 shared the highest similarity with the L. sativa Ls20ox1. The high accumulation of both HaGA20ox transcripts coincided with the period of rapid growth of the sunflower embryo, suggesting a role for GA in the first phase of embryo maturation. HaGA20ox1 mRNA was also detected in all the organs tested but occurred at a higher level in the vegetative shoot and the root, whereas HaGA20ox2 transcripts were preferentially accumulated in inflorescence meristems, vegetative shoots, internodal stem, and roots. Treatment of sunflower plants with an inhibitor of GA biosynthesis showed that HaGA20ox2, but not HaGA20ox1, was subject to feedback regulation by a reduction of bioactive GAs.

EPIPHYLLY IN A VARIANT OF HELIANTHUS ANNUUS # H. TUBEROSUS INDUCED BY IN VITRO TISSUE CULTURE

A variant clone (EMB‐2) derived by in vitro tissue culture of the interspecific hybrid Helianthus annuus × Helianthus tuberosus shows a particular deviation from the usual pattern of plant development in that it produces, both in vitro and in vivo, epiphyllous embryos and/or shootlike structures. Ectopic structures, which are usually arranged in clusters or rows along preexisting veins, originate asynchronously from epidermal cells of the adaxial surface of the leaf blade. Sometimes embryos and buds are also detected on the adaxial plane of the petioles and at the nodes of the stem. EMB‐2 individual plants differ greatly in terms of the timing and extent of phenotypic expression of epiphylly. Leaves precociously affected by ectopic structures show a more drastic alteration in the differentiation process. Growth is arrested, the spongy parenchyma and air spaces are absent, and the mesophyll cells do not enlarge. Excluding the veins and epidermis, the leaves are wholly composed of isodiametric cells that are regularly arranged in parallel rows that have dense cytoplasm and prominent nuclei. Ectopic structures isolated from leaves and cultured in vitro mostly produce plantlets with the same phenotype as the original clones. In vivo, the EMB‐2 plants are propagated by tubers. Often, the shoot‐meristems that originate from tubers exhibit a teratological appearance and die without further development. However, several normal shoots grow and produce plants that display epiphyllous structures like those of the parent plants. Alterations of the endogenous hormonal levels or mutations in genes involved in the switch from indeterminate to determinate cell fate may be responsible for the ectopic development of shoots and embryos on leaves of EMB‐2 variant.