Roberte Bronner

Regulation of Sterol Content in Membranes by Subcellular Compartmentation of Steryl-Esters Accumulating in a Sterol-Overproducing Tobacco Mutant.

The study of sterol overproduction in tissues of LAB 1–4 mutant tobacco (Nicotiana tabacum L. cv Xanthi) (P. Maillot-Vernier, H. Schaller, P. Benveniste, G. Belliard [1989] Biochem Biophys Res Commun 165: 125–130) over several generations showed that the overproduction phenotype is stable in calli, with a 10-fold stimulation of sterol content when compared with wild-type calli. However, leaves of LAB 1–4 plants obtained after two steps of self-fertilization were characterized by a mere 3-fold stimulation, whereas calli obtained from these plants retained a typical sterol-overproducing mutant phenotype (i.e. a 10-fold increase of sterol content). These results suggest that the expression of the LAB 1–4 phenotype is dependent on the differentiation state of cells. Most of the sterols accumulating in the mutant tissues were present as steryl-esters, which were minor species in wild-type tissues. Subcellular fractionation showed that in both mutant and wild-type tissues, free sterols were associated mainly with microsomal membranes. In contrast, the bulk of steryl-esters present in mutant tissues was found in the soluble fraction of cells. Numerous lipid droplets were detected in the hyaloplasm of LAB 1–4 cells by cytochemical and cytological techniques. After isolation, these lipid granules were shown to contain steryl-esters. These results show that the overproduced sterols of mutant tissues accumulate as steryl-esters in hyaloplasmic bodies. The esterification process thus allows regulation of the amount of free sterols in membranes by subcellular compartmentation.

Immuno-cytochemical localization of indole-3-acetic acid during induction of somatic embryogenesis in cultured sunflower embryos

Abstract. Immature zygotic embryos of sunflower (Helianthus annuus L.) produce somatic embryos when cultured on medium supplemented with a cytokinin as the sole source of exogenous growth regulators. The timing of the induction phase and subsequent morphogenic events have been well characterized in previous work. We address here the question of the role of endogenous indole-3-acetic acid (IAA), since auxins are known to have a crucial role in the induction of somatic embryogenesis in many other culture and regeneration systems. The fact that in the sunflower system no exogenous auxin is required for the induction of somatic embryos makes this system very suitable for the study of the internal dynamics of IAA. We used an immuno-cytochemical approach to visualize IAA distribution within the explants before, during and after the induction phase. IAA accumulated transiently throughout cultured embryos during the induction phase. The detected signal was not uniform but certain tissues, such as the root cap and the root meristem, accumulated IAA in a more pronounced manner. IAA accumulation was not restricted to the reactive zone but the kinetics of endogenous variations strikingly mimic the pulse of IAA that is usually provoked by exogenous IAA application. The direct evidence presented here indicates that an endogenous auxin pulse is indeed among the first signals leading to the induction of somatic embryogenesis.

Somatic embryogenesis and organogenesis induced on the immature zygotic embryo of sunflower (Helianthus annum L.) cultivated in vitro: role of the sugar.

SummaryImmature zygotic embryos of sunflower constitute an experimental system where the change of a single key factor (sucrose concentration) conditions the in vitro morphogenesis to either organogenesis (87 mM sucrose) or somatic embryogenesis (350 mM sucrose). Experiments with a variety of culture media differing in the sugar type and concentration, as well as osmotic pressure, indicate that a minimal threshold level of both, sugar supply and osmotic pressure, are required for somatic embryogenesis, but not organogenesis, to occur. The nature of the sugar used, though, was less important.

Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.) shoot apices: transformation patterns

Apical segments of embryonic axes of sunflower (Helianthus annuus L.) embryos were submitted to co-culture experiments with a disarmed strain of Agrobacterium tumefaciens, harbouring a plasmid coding for the marker enzyme β-glucuronidase. The expression patterns of this marker were analysed at different developmental stages of the regenerated shoots. The results are consistent with the hypothesis that transformed shoots originate from transformation events that have occurred within the existing meristems. Two of the resulting chimaeric plants have been analysed in detail, and some representative gene integration patterns are presented.

Transient gene expression in sunflower (Helianthus annuus L.) following microprojectile bombardment

Abstract Transient expression of the uidA gene, coding for β-glucuronidase (GUS) and driven by different promoters, has been induced in sunflower cotyledonary explants and immature zygotic embryos of different developmental stages using two particle delivery systems. Explants were evaluated for expression of β-glucuronidase activity 3 days, 2, and 4 weeks post bombardment. Immature embryos were more suitable for transformation than cotyledons derived from mature seeds. The highest level of transient GUS expression after 3 and 14 days was obtained with embryos (≤1.5 mm) precultured for 3 days in the presence of NAA and BAP. After 2 and 4 weeks of culture, increased GUS activities were determined following bombardment with plasmids harboring a doubled CaMV 35S and a stress inducible promoter, respectively. In cotyledons, GUS-expressing cells were mostly observed in the epidermal layer, while in immature embryos they were located between the epidermis and the fourth mesophyll layer. The performance of the two biolistic equipments was comparable. Under any condition, GUS expression declined with increasing culture time.

A transmitting tissue- and pollen-expressed protein from sunflower with sequence similarity to the human RTP protein

Abstract A novel pistil- and pollen-expressed gene ( sf21 ) encoding a 352 amino acid long polypeptide was isolated by differential screening of a floral cDNA library from sunflower ( Helianthus annuus L.) and characterized. The deduced polypeptide is structurally related to the human protein RTP and the mouse protein Ndr1. It also shares significant sequence homology with an unidentified polypeptide from human cerebellum (N-terminus) and with the ligand binding region of the vertebrate inositol 1,4,5 trisphosphate (IP3) receptor (C-terminus). The sf21 gene is expressed in young and mature florets of an open inflorescence but not at the floral bud stage. In the sunflower pistil, expression is restricted to the transmitting tissue and the phloem cells. The same location of expression, with an increasing concentration gradient of SF21 transcripts towards the ovule, is observed during specific stages in tobacco pistils. The transcripts gradually disappear as the pollen tubes grow through the transmitting tissue. These observations suggest that the protein acts in pollen-pistil interactions.

Expression of foreign genes in sunflower ( Helianthus annuus L.) — evaluation of three gene transfer methods

Suitable sunflower tissues and cells were transformed either by direct gene transfer into protoplasts, particle bombardment, or Agrobacterium co-culture. While all techniques allowed efficient short-term or transient expression of the introduced gene(s) in the respective tissues, stable transformation was only observed after transformation with Agrobacterium. The latter technique was suitable for the production of transgenic callus from seedling cotyledons and occasional shoots with chimaeric expression of the transgene. Detailed analysis of the interaction of Agrobacterium with this explant showed that infection efficiency was critically dependent on the co-culture conditions, and that the preferentially-transformed cells were not the ones competent for regeneration.

Formation of epiphyllous buds in sunflower (Helianthus annuus L.): induction in vitro and cellular origin

Abstract Epiphyllous buds developed on leaves of shoots derived from in vitro cultured apices of sunflower (Helianthus annuus L.) seedlings. This phenomenon was shown to be cytokinin-dependent but only occurred when the cytokinin was supplied via the cultured apices producing the leaf-bearing shoots. A 1-day exposure of apices to cytokinin was sufficient for epiphyllous bud induction, although an exposure of more than 4 days yielded maximal shoot development frequencies from these buds. Preculture of complete donor seedlings on cytokinin-supplemented media and subsequent culture of excised leaves on hormone-free medium was insufficient to induce epiphyllous bud formation. Buds became macroscopically visible after 9–11 days. At this time, epiphyllous buds consisted of a functional meristem producing true shoots which were capable of seed production. The meristems were formed by a multicellular process originating from subepidermal layers on the adaxial leaf side. The role of cytokinin and other potential factors in the induction of epiphyllous buds on sunflower leaves is discussed.

Amyloplasts as a possible indicator of morphogenic potential in sunflower protoplasts

Sunflower plants (inbred line 47 302 bcd) can be regenerated from hypocotyl protoplasts. During the regeneration process, we have observed two kinds of calli differing by their cell types. Morphogenic calli, from which regeneration of shoots was possible, were characterized by the presence of starch granules, and clusters of small meristematic-like cells. Non-morphogenic calli did not contain either meristematic-like cells or starch, but a varying proportion of the typical callus cells at more or less advanced stages of degeneration. We have analyzed samples from each step of protoplast culture for the presence of starch, i.e. the initial protoplast population, micro- and macro-colonies and calli. On each step, only a small percentage of the structures contained starch. These results are discussed in view of a possible origin of these morphogenic calli from particular cell types existing in the donor tissue, i.e. stomatal guard cells and endodermis cells.

A flower-specific gene family whose expression is regulated temporally and spatially during flower development in sunflower☆

Abstract One genomic (SF15G) and four different cDNA clones (SF15-49, SF15-2, SF15-3 and SF15-45) expressed in the young green closed sunflower ( Helianthus annuus L.) inflorescence were isolated and characterized. Northern blot analysis revealed temporal regulation of the sf15 gene family: the transcripts accumulate transiently between stage 1 and 4 of floret development and disappear before the florets reach maturity. The SF15 mRNAs were undetectable in male-sterile sunflower plants. In situ hybridization experiments showed that expression of the SF15 transcripts occurs in the anther epidermal cell layer as well as in immature pollen, in the corollar tissue, and in the papillar cells of the stigma. The sf15 gene encodes a putative polypeptide of 280 amino acids with a molecular weight of 31.7 kDa. The five sunflower SF15 proteins are highly homologous to each other (82–99% nucleotide and amino acid sequence identity). They are basic (isoelectric point of 9.6) and have a potential N-terminal signal peptide, indicating that they may enter the secretory pathway. The presence of a short hydrophobic C-terminal domain in the protein encoded by the genomic sequence but not in the proteins encoded by the cDNAs suggests an extracellular as well as a vacuolar location for this family of proteins. Southern blot analysis shows that in sunflower the sf15 gene exists as a family of genes with approximately six to eight family members. The sf15 gene is also present in the maize, tomato and cabbage genomes.