Mariangela Salvini

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.

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.

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.

Effect of copper on callus growth and gene expression of in vitro-cultured pith explants of Nicotiana glauca

Abstract Copper is a vital component of electron transfer reactions mediated by proteins such as superoxide dismutase, cytochrome c oxidase and plastocyanin, but its concentrations in the cells needs to be maintained at low levels. In fact, the same ability of this essential metal ion to transfer electrons can also make it toxic to cells when present in excess. In vitro cultured explants of Nicotiana have been extensively used as a model to analyse metal-DNA interactions. In this report, we examined the effect of copper (1, 10 and 100 μM CuSO4) on callus growth and protein synthesis of in vitro-cultured pith explants of Nicotiana glauca. In addition, a N. glauca cDNA library from Cu-treated (100 μM CuSO4) pith explants cultured in vitro for 24 h was analysed by mRNA differential screening. The copper treatments inhibited callus growth of pith explants. The extent of inhibition was directly correlated to metal concentration. One and 10 μM CuSO4 induced a notable increase of proteins synthesis relative to control explants. By contrast, 100 μM CuSO4 inhibited protein synthesis relative to control extracts. The SDS-PAGE fluorography of pith proteins revealed, in Cu-treated extracts qualitative and/or quantitative differences in the synthesis of some polypeptides compared with control explants. Copper-modulated patterns of gene expression were also analysed by mRNA differential screening. The N. glauca genes isolated from Cu-treated pith explants shared common identities with other genes known to be elicited by diverse stresses, including pathogenesis and abiotic stress. In particular, the cDNAs were homologues to genes encoding cell wall proteins (i.e., extensin, and arabinogalactan-protein) and pathogenesis-related proteins (i.e., osmotin, endochitinase and a member of the Systemic Acquired Resistance gene family). In addition, an MD-2-related lipid-recognition (ML) domain protein and the enzyme S-adenosyl-L-homocysteine (AdoHcy) hydrolase appeared involved in the response to copper stress. In animal cells, AdoHcy hydrolase is a copper binding protein in vivo, which suggests that, also in plant tissues, this enzyme may play an important role in regulating the levels and intracellular distribution of copper.

Transposon-dependent induction of Vincent van Gogh's sunflowers: exceptions revealed.

The radiate sunflower inflorescence is composed by zygomorphic ray flowers and actinomorphic disk flowers. Studies performed on mutants identify HaCYC2c, a CYCLOIDEA (CYC)‐like gene, as one of the key players controlling flower symmetry in sunflower. turf and tub mutants are characterized by a shift from zygomorphic to actinomorphic ray flowers, caused by insertion of transposable elements (TEs) in HaCYC2c gene. In dbl or Chry mutants, an insertion upstream the coding region of HaCYC2c causes the ectopic expression of the gene and the shift from actinomorphic to zygomorphic disk flowers. We focused on Chry2 mutant: a 1034 bp insertion placed 558 bp before the start codon of HaCYC2c was identified. The insertion is a truncated version of a CACTA TE. Unexpectedly, phenotypic and genetic co‐segregation analysis in F2 and F3 progenies derived from the crosses Chry2 × turf and turf × Chry2 demonstrated that CACTA insertion is not always sufficient to alter the expression of HaCYC2c gene and generate Chry2 phenotype. F3 plants homozygous for the CACTA insertion displayed either HaCYC2c transcription pattern identical to wild‐type plants or a normal heterogamous inflorescence. Stated these results, we conclude that a much more complex regulatory system stays behind the Chry2 phenotype. genesis 52:315–327, 2014.

Phytoene accumulation in sunflower decreases the transcript levels of the phytoene synthase gene

The non dormant-1 (nd-1) mutant of sunflower (Helianthus annuus L.) and fluridone-treated plants were used to investigate the effect of phytoene accumulation on the transcript levels of the phytoene synthase (Ha-PSY) gene. A deficiency at the gene coding for ζ-carotene desaturase (ZDS) characterises the nd-1 mutant, which displays an accumulation of ζ-carotene, phytofluene and cis-phytoene and the absence of β-carotene and xanthophylls. The transcript levels of Ha-PSY, in concomitance with phytoene accumulation, decreased (1.9-fold) in fluridone-treated wild-type seedlings with respect to untreated leaves. Phytoene-accumulating cotyledons of nd-1 seedlings also displayed a 2.9-fold decrease of Ha-PSY mRNA levels in comparison with the control samples. To exclude that the reduced transcript levels of Ha-PSY was dependent by a decrease of the total carotenoid content another pigment-deficient mutant, named xantha1 (xan1), was used. The xan1 mutant has a normal carotenoid complement but photodestruction of both chlorophyll a and β-carotene directly correlate with light intensity. The increase of Ha-PSY mRNA, in normal-pigmented cotyledons, was concurrent with the enhanced light intensity. The same up-regulation for Ha-PSY was observed in xan1, irrespective of the drastic reduction of the total carotenoid content displayed by mutant seedlings grown under high light intensity. Taken together, our data suggest that in H. annuus the steady state levels of Ha-PSY mRNA was negatively affects by phytoene accumulation but not by a decrease in total carotenoid content.

Molecular analysis of a sunflower gene encoding an homologous of the B subunit of a CAAT binding factor

A genomic DNA fragment containing the complete LEAFY COTYLEDON1-LIKE (HaL1L) gene was retrieved by chromosome walking. Its sequence was confirmed and elongated by screening a sunflower genomic DNA BAC Library. HaL1L, whose cDNA had already been sequenced and characterized, encodes a NF-YB subunit of a CCAAT box-binding factor (NF-Y) involved in the early stages of zygotic and somatic embryogenesis in the Helianthus genus. In the HaL1L 5′-flanking region, elements specific to a putative TATA-box promoter and two “CG isles” were identified. An investigation of the methylation status of these CG rich DNA regions showed that differentially methylated cytosines were recognizable in the DNA of embryos on the fifth day after pollination in comparison to leaf DNA suggesting that during plant development epigenetic regulation of HaL1L transcription was achieved by methylating cytosine residues. We also searched the HaL1L nucleotide sequence for cis-regulatory elements able to interact with other transcription factors (TFs) involved in the HaL1L regulation. Of the elements identified, one of the most intriguing is WUSATA, the target sequence for the WUSCHEL (WUS) TF, which may be part of a complex regulation network controlling embryo development. In this article, we show that the WUSATA target site, located in the intron of HaL1L, is able to bind the TF WUS. Interestingly, we found auxin and abscisic acid responsive motifs in the HaL1L promoter region suggesting that this gene may additionally by under hormonal control. Finally, the presence of a cytoplasmic polyadenylation signal downstream to the coding region indicates that this gene may also be controlled at the translation level by a temporarily making the pre-synthesized HaL1L mRNA unavailable for protein synthesis.

Excisions of a defective transposable CACTA element (Tetu1) generate new alleles of a CYCLOIDEA-like gene of Helianthus annuus

Tubular ray flower (turf) is a sunflower mutant that caught attention because it bears actinomorphic ray flowers, due to the presence of an active, although non-autonomous CACTA transposon (Tetu1) in the TCP domain of a CYCLOIDEA-like gene, HaCYC2c, a major regulator of sunflower floral symmetry. Here, we analyzed its excision rates in F3 population deriving from independent crosses of turf with common sunflower accessions. Our results suggest that the excision rate, ranging from 1.21 to 6.29%, depends on genetic background; moreover, the absence of somatic sectors in inflorescences of revertant individuals analyzed (182) and genetic analyses suggests a tight developmental control of Tetu1 excision, likely restricted to germinal cells. We individuate events of Tetu1 excision through molecular analysis that restore the wild type (WT) HaCYC2c allele, but even transposon excisions during which footprints are left. All mutations we detected occurred at the TCP basic motif and cause a change in ray flower phenotype. In particular, we selected five mutants with a one-to-four amino acid change that influence the capacity of reproductive organ development and ray flower corolla shaping (MUT-1, -2, -3, -4, -5). Revertant alleles not affecting turf phenotype (i.e. reading frame mutations) have also been identified (MUT-6). In all mutants, Real-time quantitative PCR (qPCR) experiments revealed variations of the steady state level of HaCYC2c mRNA. MUT-1 and MUT-4 showed a significant HaCYC2c down-regulation with respect to WT. A large variation within the biological replicates of MUT-2, MUT-3 and MUT-5 was detected and not significant differences in transcription levels between mutants and WT were observed. We detected low steady state level of HaCYC2c mRNA both in turf as in MUT-6. A three dimensional (3D) structure prediction tool let us predict an incorrect folding of the TCP protein already after a single amino acid deletion. This in turn is detectable as the restore of traits that are not peculiar of WT ray flowers, such as male fertility. Our analysis of an active TE sheds light on the TCP motif of the HaCYC2c gene and suggests that Tetu1 may be useful to obtain new natural mutants and for transposon tagging in different inbred lines of sunflower.

Alpha-tryptophan synthase of Isatis tinctoria: gene cloning and expression

Indole producing reaction is a crux in the regulation of metabolite flow through the pathways and the coordination of primary and secondary product biosynthesis in plants. Indole is yielded transiently from indole-3-glycerol phosphate and immediately condensed with serine to give tryptophan, by the enzyme tryptophan synthase (TS). There is evidence that plant TS, like the bacterial complex, functions as an alpha beta heteromer. In few species, e.g. maize, are known enzymes, related with the TS alpha-subunit (TSA), able to catalyse reaction producing indole, which is free to enter the secondary metabolite pathways. In this contest, we searched for TSA and TSA related genes in Isatis tinctoria, a species producing the natural blue dye indigo. The It-TSA cDNA and the full-length exons/introns genomic region were isolated. The phylogenetic analysis indicates that It-TSA is more closely related to Arabidopsis thaliana At-T14E10.210 TSA (95.7% identity at the amino acid level) with respect to A. thaliana At-T10P11.11 TSA1-like (63%), Zea mays indole-3-glycerol phosphate lyase (54%), Z. mays TSA (53%), and Z. mays indole synthase (50%). The It-TSA cDNA was also able to complement an Escherichia coli trpA mutant. To examine the involvement of It-TSA in the biosynthesis of secondary metabolism compounds, It-TSA expression was tested in seedling grown under different light conditions. Semi-quantitative RT-PCR showed an increase in the steady-state level of It-TSA mRNA, paralleled by an increase of indigo and its precursor isatan B. Our results appear to indicate an involvement for It-TSA in indigo precursor synthesis and/or tryptophan biosynthesis.