An analysis of sequence variability in eight genes putatively involved in drought response in sunflower (Helianthus annuus L.).

With the aim to study variability in genes involved in ecological adaptations, we have analysed sequence polymorphisms of eight unique genes putatively involved in drought response by isolation and analysis of allelic sequences in eight inbred lines of sunflower of different origin and phenotypic characters and showing different drought response in terms of leaf relative water content (RWC). First, gene sequences were amplified by PCR on genomic DNA from a highly inbred line and their products were directly sequenced. In the absence of single nucleotide polymorphisms, the gene was considered as unique. Then, the same PCR reaction was performed on genomic DNAs of eight inbred lines to isolate allelic variants to be compared. The eight selected genes encode a dehydrin, a heat shock protein, a non-specific lipid transfer protein, a z-carotene desaturase, a drought-responsive-element-binding protein, a NAC-domain transcription regulator, an auxin-binding protein, and an ABA responsive-C5 protein. Nucleotide diversity per synonymous and non-synonymous sites was calculated for each gene sequence. The π (a)/π (s) ratio range was usually very low, indicating strong purifying selection, though with locus-to-locus differences. As far as non-coding regions, the intron showed a larger variability than the other regions only in the case of the dehydrin gene. In the other genes tested, in which one or more introns occur, variability in the introns was similar or even lower than in the other regions. On the contrary, 3'-UTRs were usually more variable than the coding regions. Linkage disequilibrium in the selected genes decayed on average within 1,000 bp, with large variation among genes. A pairwise comparison between genetic distances calculated on the eight genes and the difference in RWC showed a significant correlation in the first phases of drought stress. The results are discussed in relation to the function of analysed genes, i.e. involved in gene regulation and signal transduction, or encoding enzymes and defence proteins.

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

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.

Ligulate inflorescence of Helianthus × multiflorus, cv. Soleil d'Or, correlates with a mis-regulation of a CYCLOIDEA gene characterised by insertion of a transposable element.

Members of CYCLOIDEA (CYC)/TEOSINTE BRANCHED1 (TB1) transcription factor family are essential to control flower symmetry and inflorescence architecture. In the Helianthus annuus genome, ten CYC/TB1 genes have been identified. Studies performed on mutants recognised HaCYC2c as one of the key players controlling zygomorphism in sunflower. We identified CYC2c genes in the diploid Helianthus decapetalus (HdCYC2c) and in the interspecific hybrid Helianthus × multiflorus (H × mCYC2cA and H × mCYC2cB), a triploid (2n = 3× = 51), originated from unreduced eggs of H. decapetalus fertilised by reduced H. annuus male gametes. Phylogenetic analysis showed that HdCYC2c and H × mCYC2c were placed within a CYC2 subclade together with HaCYC2c but distinct from it. The present data showed that in H. × multiflorus the allele derived from H. annuus is deleted or highly modified. The H. × multiflorus taxon exists as radiate and ligulate inflorescence types. We analysed CYC2c expression in H. decapetalus and in the cultivar 'Soleil d'Or' of H. × multiflorus, a ligulate inflorescence type with actinomorphic corolla of disk flowers transformed into a zygomorphic ray-like corolla. In H. decapetalus, the HdCYC2c gene showed differential expression between developing flower types, being up-regulated in the corolla of ray flowers in comparison to the disk flower corolla. In H. × multiflorus, an insertion of 865 bp, which is part of a CACTA transposable element, was found in the 5'-untranslated region (5'-UTR) of H × mCYC2cB. This insertion could promote, even with epigenetic mechanisms, ectopic expression of the gene throughout the inflorescence, resulting in the observed loss of actinomorphy and originating a ligulate head.

A GRAS-like gene of sunflower (Helianthus annuus L.) alters the gibberellin content and axillary meristem outgrowth in transgenic Arabidopsis plants.

The GRAS proteins belong to a plant transcriptional regulator family that function in the regulation of plant growth and development. Despite their important roles, in sunflower only one GRAS gene (HaDella1) with the DELLA domain has been reported. Here, we provide a functional characterisation of a GRAS-like gene from Helianthus annuus (Ha-GRASL) lacking the DELLA motif. The Ha-GRASL gene contains an intronless open reading frame of 1,743 bp encoding 580 amino acids. Conserved motifs in the GRAS domain are detected, including VHIID, PFYRE, SAW and two LHR motifs. Within the VHII motif, the P-H-N-D-Q-L residues are entirely maintained. Phylogenetic analysis reveals that Ha-GRASL belongs to the SCARECROW LIKE4/7 (SCL4/7) subfamily of the GRAS consensus tree. Accumulation of Ha-GRASL mRNA at the adaxial boundaries from P6/P7 leaf primordia suggests a role of Ha-GRASL in the initiation of median and basal axillary meristems (AMs) of sunflower. When Ha-GRASL is over-expressed in Arabidopsis wild-type plants, the number of lateral bolts increases differently from untransformed plants. However, Ha-GRASL slightly affects the lateral suppressor (las-4-) mutation. Therefore, we hypothesise that Ha-GRASL and LAS are not functionally equivalent. The over-expression of Ha-GRASL reduces metabolic flow of gibberellins (GAs) in Arabidopsis and this modification could be relevant in AM development. Phylogenetic analysis includes LAS and SCL4/7 in the same major clade, suggesting a more recent separation of these genes with respect to other GRAS members. We propose that some features of their ancestor, as well as AM initiation and outgrowth, are partially retained in both LAS and SCL4/7.

Orange, yellow and white-cream: inheritance of carotenoid-based colour in sunflower pollen.

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.

Expression of HtKNOT1, a class I KNOX gene, overlaps cell layers and development compartments of differentiating cells in stems and flowers of Helianthus tuberosus.

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.

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.

Origin and development in vitro of shoot buds and somatic embryos from intact roots of Helianthus annuus x H. tuberosus.

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.

Hormonal influence on photocontrol of the protandry in the genus Helianthus.

Under natural photoperiodic conditions protandry in hermaphrodite disc flowers of sunflower (Helianthus annuus L.) is determined by the different elongation rates of the style and filaments. The elongation of the filament and style starts simultaneously after the daily dark period, but the style growth rate is slower. When plants close to anthesis are exposed to continuous white light (WL) a loss of protandry occurs: the filaments do not grow far enough to extrude the anthers from the corolla. The histological analyses show that the number of filament epidermal cells remains unaltered after organ elongation and that cells respond to photoperiod only by cell expansion. Emasculation does not substantially inhibit filament cell expansion, whereas isolation of the filament or stamen from the corolla suggests that this organ could be the perception site of the filament growth stimulus. In vitro treatments with auxin (indole-3-acetic acid, IAA or alpha-naphthaleneacetic acid, NAA) reverses the inhibition of cell expansion caused by continuous WL, whereas gibberellic acid (GA(3)) at high concentrations reproduces the effect of continuous WL. Experiments carried out on various Helianthus spp. show that all these plants have evolved the same photo- and hormonal-control of the protandry. In experiments in which the light treatments were continued for 24 h, the auxins drastically reduced the inhibiting effect of red light (R) and dichromatic treatments FR (far red)+R, whereas GA(3) repressed filament extension regardless of light quality. As far as auxins are concerned, the response of sunflower filaments does not appear to be connected with the polar transport of the hormone. Moreover, the promoting effect of darkness is not mediated by an increase of endogenous free IAA in disc flowers. However, sunflower filaments manifested a similar temporal pattern of response to the light/dark cycle and to auxin.

Heterochromatin and repetitive DNA frequency variation in regenerated plants of Helianthus annuus L.

Plant regeneration from cotyledons of seeds of a single progeny of a pure line of Helianthus annuus was studied in respect of the nuclear DNA contents of control and regenerated plants. Control plants were divided into two groups: those developed from seeds at the periphery of the inflorescence (showing a high basic 4C DNA content) and those from seeds developed in the middle of the inflorescence (showing a low basic 4C DNA content). It was observed that plants from peripheral seeds have a higher morphogenetic potential than those from central seeds. Cytophotometric analyses indicated that plants regenerated from cotyledons of both peripheral and central seeds show the same basic 4C DNA amount, which is higher that that observed in vivo in peripheral seeds. Molecular analysis by slot blotting and hybridization with different DNA families showed that the difference in nuclear DNA content between plants from peripheral and central seeds in vivo are mainly related to differences in the frequency of highly repeated, "slow" medium repeated (MR2), and ribosomal DNA families; by contrast, the increase in DNA amount in regenerated plants is mainly due to "fast" medium repeated sequences (MR1). Moreover, the frequency of kinetically isolated "unique" sequences was higher in peripheral seeds than in central ones and still higher in regenerated plants. Optical-density measurements of interphase nuclei showed an increase of heterochromatin in regenerated plants, suggesting that, whatever DNA is amplified in these plants, it remains condensed and probably inactive.

Epiphylly in a Variant of Helianthus Annuus x 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 x 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.

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 (HaDhnla) 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.