[Show abstract][Hide abstract] ABSTRACT: Inheritance of pollen colour was studied in sunflower (Helianthus annuus L.) using three distinct pollen colour morphs: orange, yellow and white-cream. Orange is the most common colour of sunflower pollen, while the yellow morph is less frequent. These two types were observed in the inbred lines F11 and EF2L, respectively. White-cream pollen is a rare phenotype in nature, and was identified in a mutant, named white-cream pollen, recovered in the R(2) generation of an in vitro regenerated plant. The F11 inbred line was used as starting material for in vitro regeneration. The carotenoid content of these three pollen morphs differed, and was extremely reduced in white-cream pollen. The phenotype of F(1) populations obtained by reciprocal crosses revealed that the orange trait was dominant over both white-cream and yellow. Segregation of F(2) populations of both crosses, orange x yellow and orange x white-cream, approached a 3:1 ratio, indicating the possibility of simple genetic control. By contrast, a complementation cross between the two lines with white-cream and yellow pollen produced F(1) plants with orange pollen. The F(2) populations of this cross-segregated as nine orange: four white-cream: four yellow. A model conforming to the involvement of two unlinked genes, here designated Y and O, can explain these results. Accessions with yellow pollen would have the genotype YYoo, the white-cream pollen mutant would have yyOO and the accession with orange pollen would have YYOO. Within F(2) populations of the cross white-cream x yellow a new genotype, yyoo, with white-cream pollen was scored. The results of the cross yyoo x YYoo produced only F(1) plants with yellow pollen, supporting a recessive epistatic model of inheritance between two loci. In this model, yy is epistatic on O and o. In F(2) populations, the distributions of phenotypic classes suggested that the genetic control of carotenoid content is governed by major genes, with large effects segregating in a background of polygenic variation. These three pollen morphs can provide insight into the sequence in which genes act, as well into the biochemical pathway controlling carotenoid biosynthesis in anthers and the transfer of these different pigments into pollenkitt.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
Annals of Botany 02/2009; 103(5):735-47. DOI:10.1093/aob/mcn266 · 3.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gibberellin (GA) signalling pathways are prime targets for producing dwarf high-yielding crop varieties. The GA 2-oxidases catalyze the 2β-hydroxylation of several biologically active GAs, leading to the homeostatic regulation of their endogenous level. A gene, HaGA2ox1, and its corresponding cDNA that contains all the features and exhibits homology to GA 2-oxidases of several plant species has been isolated from vegetative shoots of sunflower (Helianthus annuus). Sequence analysis revealed that HaGA2ox1 consists of three exons and two introns. The predicted amino acid sequence of HaGA2ox1 shares the highest similarities (89% of identity) with the GA 2-oxidase of Lactuca sativa (LsGA2ox1). The phylogenetic analysis divides the large GA 2-oxidase family into three separate classes. The amino acid sequence of HaGA2ox1, included in the class I, predicts that it would be able to catabolize C 19-GAs, including bioactive GAs, as GA 1 , and its immediate precursor, GA 20. Within the class I clade, HaGA2ox1 formed a sub-clade with LsGA2ox1 and GA 2-oxidase of Chrysanthemum x morifolium (CmGA2ox1). HaGA2ox1 is ubiquitously expressed in sunflower organs but most abundantly in the inflorescence meristem and in the roots. Treatments of sunflower seeds with gibberellic acid showed that HaGA2ox1 was subject to a slight positive feed-forward regulation by an increase of bioactive GAs. The HaGA2ox1 is a candidate gene to manipulate the amount of bioactive GAs in sunflower by a genetic engineering approach.
Plant Breeding, Edited by Niko Huttunen and Taavi Sinisalo, 01/2009: chapter 9: pages 275-291; Nova Science Publishers, Inc.., ISBN: ISBN: 978-1-60741-624-1
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: KNOTTED1-like homeobox (KNOX) genes act in the shoot apical meristem (SAM) to provide an uncommitted population of cells for organogenic programs. In addition to playing a role in SAM function and leaf morphology, there is also evidence of the involvement of KNOX genes in some features of flower development. The expression pattern of HtKNOT1, a class I KNOX gene, in inflorescence meristems, floral meristems and floral organs of two related species, Helianthus tuberosus and H. annuus strengthens this view. HtKNOT1 mRNAs have been detected in the inflorescence and floral meristems as well within lateral organ primordia (i.e. floral bracts, petals, stamens and carpels). In more differentiated flowers, the expression of HtKNOT1 was restricted to developing ovules and pollen. Class I KNOX genes may play a dual role being required to maintain the meristem initials as well as initiating differentiation and/or conferring new cell identity. In the genus Helianthus, some KNOX genes could cooperate at the floral level with additional factors that more specifically control floral organs and pollen development.
Floriculture, Ornamental and Plant Biotechnology Volume V, Edited by Jaime A. Teixeira da Silva, 01/2008: chapter 32: pages 305-310; Global Science Books, UK., ISBN: ISBN: 978-4-903313-12-2
[Show abstract][Hide abstract] ABSTRACT: In plant, post-embryonic development relies on the activities of indeterminate cell populations termed meristems, spatially clustered cell lineages, wherein a subset divides indeterminately. For correct growth, the plant must maintain a constant flow of cells through the meristem, where the input of dividing pluripotent cells offsets the output of differentiating cells. KNOTTED1-like homeobox (KNOX) genes are expressed in specific patterns in the plant meristems and play important roles in maintaining meristematic cell identity. We have analyzed the expression pattern of HtKNOT1, a class I KNOX gene of Helianthus tuberosus, in stems, inflorescence meristems, floral meristems and floral organs. HtKNOT1 is expressed in cambial cells, phloem cells and xylematic parenchyma within apical stem internodes, while in basal internodes HtKNOT1 expression was restricted to the presumptive initials and recently derived phloem cells. In the reproductive phase, HtKNOT1 mRNAs were detected in both the inflorescence and floral meristems as well within lateral organ primordia (i.e. floral bracts, petals, stamens and carpels). In more differentiated flowers, the expression of HtKNOT1 was restricted to developing ovules and pollen mother cells. HtKNOT1 may play a dual role being required to maintain the meristem initials as well as initiating differentiation and/or conferring new cell identity. In particular, it is possible that HtKNOT1 cooperates at floral level with additional factors that more specifically control floral organs and pollen development in H. tuberosus.
Cell Biology International 11/2007; 31(10):1280-7. DOI:10.1016/j.cellbi.2007.03.031 · 1.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The tubular ray flower (turf) mutant of sunflower (Helianthus annuus L.) is characterized by a change from a zygomorphic corolla to a nearly actinomorphic tubular-like corolla of ray flowers that also achieves the ability to differentiate fertile stamens and ovules. The recessive turf mutation is found to be unstable, spontaneously reverting to a wild-type or nearly wild-type phenotype. Reversion frequencies were 1.70% in the original inbred line R1634 (where turf was first identified), 0.17% and 3.20% in two turf/turf homozygous F3 progenies, derived from crossing a turf mutant with two different inbred lines. As no chimeric plant was found it is likely that the reversion had occurred either in the germ line or within the zygote. Self-pollinated wild-type reverted plants segregated into normal and mutant phenotypes fitting a monogenic 3 : 1 ratio, thus indicating that the reversion affected a single allele. The hypothesis of an epigenetic mechanism is discussed.
[Show abstract][Hide abstract] ABSTRACT: Plant lateral organs such as leaves arise from a group of initial cells within the flanks of the shoot apical meristem (SAM). Alterations in the initiation of lateral organs are often associated with changes in the dimension and arrangement of the SAM as well as with abnormal hormonal homeostasis. A mutation named stem fasciated (stf) that affects various aspects of plant development, including SAM shape and auxin level, was characterized in sunflower (Helianthus annuus).
F1, F2 and F3 generations were obtained through reciprocal crosses between stf and normal plants. For the genetic analysis, a chi2 test was used. Phenotypic observations were made in field-grown and potted plants. A histological analysis of SAM, hypocotyl, epicotyl, stem and root apical meristem was also conducted. To evaluate the level of endogenous indole-3-acetic acid (IAA), a capillary gas chromatography-mass spectrometry-selected ion monitoring analysis was performed.
stf is controlled by a single nuclear recessive gene. stf plants are characterized by a dramatically increased number of leaves and vascular bundles in the stem, as well as by a shortened plastochron and an altered phyllotaxis pattern. By histological analysis, it was demonstrated that the stf phenotype is related to an enlarged vegetative SAM. Microscopy analysis of the mutant's apex also revealed an abnormal enlargement of nuclei in both central and peripheral zones and a disorganized distribution of cells in the L2 layer of the central zone. The stf mutant showed a high endogenous free IAA level, whereas auxin perception appeared normal.
The observed phenotype and the high level of auxin detected in stf plants suggest that the STF gene is necessary for the proper initiation of primordia and for the establishment of a phyllotactic pattern through control of both SAM arrangement and hormonal homeostasis.
Annals of Botany 11/2006; 98(4):715-30. DOI:10.1093/aob/mcl153 · 3.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Helianthus annuus LEAFY 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 x 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.
Development Genes and Evolution 06/2006; 216(5):253-64. DOI:10.1007/s00427-005-0050-7 · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epiphylly, occurring in a somaclonal variant (EMB-2) of the interspecific hybrid Helianthus annuus x H. tuberosus, was used to investigate molecular and cyto-physiological mechanisms that underlie cellular fate change. EMB-2 plants are characterized by profuse proliferation of shoot- and embryo-like structures on some leaves. We addressed the putative relationship between cytokinins and knox genes in EMB-2 plants. A class I knox gene, HtKNOT1, was isolated from H. tuberosus. A high level of HtKNOT1 transcripts was detected in EMB-2 epiphyllous leaves compared to non-epiphyllous (NEP) ones. In addition, epiphylly was related to a localized increases in zeatin and N-glycosylated cytokinins. As ectopic morphogenesis proceeded, HtKNOT1 transcripts and zeatin co-localized and showed different patterns in ectopic shoot compared with embryo-like structures, consistent with the differential role of both cytokinin and knox genes in the two morphogenetic events. Notably, a massive shoot/embryo regeneration was induced in EMB-2 NEP leaves by in vitro zeatin treatment. These results clearly indicate that localized cytokinin accumulation and ectopic expression of HtKNOT1 are closely linked in the epiphylly of EMB-2 plants.
[Show abstract][Hide abstract] ABSTRACT: The inflorescence of sunflower (Helianthus annuus L.) is heterogamous with zygomorphic ray flowers located in the outermost whorl of the head and actinomorphic disk flowers arrayed in arcs radiating from the center of the head. The ray flowers are sterile with large flat ovaries with no ovules; disk flowers are hermaphrodite, carrying both male and female organs. Two mutants with altered corolla symmetry are known in sunflower: the first, named Chrysanthemoides (Chry) is characterized by a shift from the polysymmetric corolla of disk flowers into a monosymmetric-like corolla; the second, named tubular ray flower (turf), shows a shift from the zygomorphic corolla of ray flowers into a nearly actinomorphic tubular-like corolla. We report in detail the morphological floral features of Chry and turf, demonstrating that both mutations also affect the development of stamens and carpels. Most disk flowers found in the peripheral whorls of Chry heads showed drastic reduction in stamen length, as well as the absence of ovules, and developed an unbranched style. In contrast, tubular-like ray flowers of turf achieved the ability to differentiate both fertile stamens and ovules. Homeotic transformations were also identified in the tubular-like ray flowers of turf, affecting both filaments and anthers that displayed petaloid-like traits. Notably, the turf mutation was not completely stable, occasionally reverting to nearly " wild-type " phenotype. Our results point to a primary role for TURF and CHRY in the programming of the corolla symmetry and suggest a key interaction of both genes with floral organ identity genes.
Floriculture, Ornamental and Plant Biotechnology Volume 1, Edited by Jaime A. Teixeira da Silva, 01/2006: chapter 7: pages 61-70; Global Science Books, UK., ISBN: ISBN: 978-4-903313-00-9
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: A relative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) approach was performed to assess ζ-carotene desaturase (HaZds) gene expression in sunflower (Helianthus annuus L.). HaZds was highly expressed in cotyledons and in young and mature leaves, but transcript levels were comparatively lower in both stems and roots. The HaZds transcript levels were influenced by leaf expansion, which suggests that HaZds expression is regulated during leaf development. The key role of HaZds in controlling carotenoid biosynthesis is indicated by the concurrent increase of HaZds transcript levels with the light-dependent enhanced carotenoid production in cotyledons of sunflower.
[Show abstract][Hide abstract] ABSTRACT: The non dormant-1 (nd-1) mutant of sunflower (Helianthus annuus L.) is characterized by an albino and viviparous phenotype. Pigment analysis by spectrophotometer and HPLC demonstrated in nd-1 cotyledons the absence of beta-carotene, lutein and violaxanthin. Additionally, we found a strong accumulation of zeta-carotene and, to a lesser extent, of phytofluene and cis-phytoene in nd-1 seedlings grown in very dim light (1 micro mol m(-2) s(-1)). These results suggested that zeta-carotene desaturation was impaired in the mutant plants. To understand the molecular basis of the nd-1 mutation, we cloned and characterized the zeta-carotene desaturase (Zds) gene from sunflower. A reconstructed full-length sequence (1,916 bp) of the Zds cDNA was obtained from homozygous Nd-1/Nd-1 wild-type plants. It contains a 1,761-bp CDS, 62 nucleotides of 5'-untranslated region (UTR), and 77 nucleotides of 3'-UTR. The predicted protein (64.9 kDa) consists of 587 amino acid residues with a putative transit sequence for plastid targeting in the N-terminal region and a typical amino oxidase domain that includes the flavin adenosine dinucleotide (FAD) binding motif. The phylogenetic analysis demonstrated that the sunflower Zds was clustered to marigold (Tagetes) Zds gene, for which it showed an overall aminoacidic identity of 96.6% and resulted strictly correlated with other Zds sequences of higher plants. Interestingly, RT-PCR analyses showed that nd-1 plants were unable to accumulate Zds transcripts. Sequence information from the Zds cDNA was used to design specific primers and to isolate the full-length exons/introns region of the gene. The sunflower Zds gene (HaZds) comprises 14 exons and 13 introns scattered in a ca. 5.0-kb region. Also, HaZds showed a high conservation of the distribution and size of the exons with rice Zds gene. Based on genomic Southern analysis, the nd-1 genome disclosed a large deficiency at the Zds locus.
[Show abstract][Hide abstract] ABSTRACT: A variant clone of the tetraploid (2n = 4x = 68) interspecific hybrid Helianthus annuus x H. tuberosus derived by in vitro tissue culture showed a deviation from the usual pattern of organization of the plant body. This variant developed shoot-like structures and somatic embryos from intact adventitious roots of in vitro-grown plantlets. The morphogenetic structures were not normally able to differentiate complete plants. They did show cellular proliferation with the inception of additional secondary embryos, leaf-like structures and unorganized masses of callus. Nevertheless, some ectopic structures isolated from roots and transferred onto fresh basal medium without growth regulators were able to produce plantlets that exhibited the same phenotype as the original clone. Histological analyses demonstrate that they originate from cortical cells in association with the development of lateral root primordia.
Annals of Botany 08/2003; 92(1):145-51. DOI:10.1093/aob/mcg116 · 3.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The initiation and growth of axillary meristems are fundamental components of plant architecture. Here, we describe the mutant missing flowers (mf) of Helianthus annuus characterized by the lack of axillary shoots. Decapitation experiments and histological analysis indicate that this phenotype is the result of a defect in axillary meristem initiation. In addition to shoot branching, mutation affects floral differentiation. The indeterminate inflorescence of sunflower (capitulum) is formed of a large flat meristem which produces floret primordia in multiple spirals. In wildtype plants a bisecting crease divides each primordium in two distinct bumps that adopt different fate. The peripheral (abaxial) part of the primordium becomes a small leaf-like bract and the adaxial part becomes a flower. In the mf mutant, the formation of flowers at the axil of bracts is precluded. Histological analyses show that in floret primordia of the mutant a clear subdivision in dyads is not established. The primordia progressively bend inside and only large involucral floral bracts are developed. The results suggest that the MISSING FLOWERS gene is essential to provide or perceive an appropriate signal to the initiation of axillary meristems during both vegetative and reproductive phases.