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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.

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.

CYP94A1 is a cytochrome P450 (P450) catalyzing fatty acid (FA) omega-hydroxylation in Vicia sativa seedlings. To study the physiological role of this FA monooxygenase, we report here on its regulation at the transcriptional level (Northern blot). Transcripts of CYP94A1, as those of two other P450-dependent FA hydroxylases (CYP94A2 and CYP94A3) from V. sativa, are barely detectable during the early development of the seedlings. CYP94A1 transcripts, in contrast to those of the two other isoforms, are rapidly (less than 20 min) and strongly (more than 100 times) enhanced after treatment by clofibrate, an hypolipidemic drug in animals and an antiauxin (p-chlorophenoxyisobutyric acid) in plants, by auxins (2,4-dichlorophenoxyacetic acid and indole-3-acetic acid), by an inactive auxin analog (2,3-dichlorophenoxyacetic acid), and also by salicylic acid. All these compounds activate CYP94A1 transcription only at high concentrations (50-500 micromolar range). In parallel, these high levels of clofibrate and auxins modify seedling growth and development. Therefore, the expression of CYP94A1 under these conditions and the concomitant morphological and cytological modifications would suggest the implication of this P450 in a process of plant defense against chemical injury.

CYP94A1 is a cytochrome P450 (P450) catalyzing fatty acid (FA) ω-hydroxylation in Vicia sativa seedlings. To study the physiological role of this FA monooxygenase, we report here on its regulation at the transcriptional level (Northern blot). Transcripts of CYP94A1, as those of two other P450-dependent FA hydroxylases (CYP94A2 and CYP94A3) from V. sativa, are barely detectable during the early development of the seedlings. CYP94A1 transcripts, in contrast to those of the two other isoforms, are rapidly (less than 20 min) and strongly (more than 100 times) enhanced after treatment by clofibrate, an hypolipidemic drug in animals and an antiauxin (p-chlorophenoxyisobutyric acid) in plants, by auxins (2,4-dichlorophenoxyacetic acid and indole-3-acetic acid), by an inactive auxin analog (2,3-dichlorophenoxyacetic acid), and also by salicylic acid. All these compounds activate CYP94A1 transcription only at high concentrations (50-500 μM range). In parallel, these high levels of clofibrate and auxins modify seedling growth and development. Therefore, the expression of CYP94A1 under these conditions and the concomitant morphological and cytological modifications would suggest the implication of this P450 in a process of plant defense against chemical injury.[PUBLICATION ABSTRACT]

LIM proteins are important eucaryotic developmental regulators characterized by the presence of one or several double zinc finger motifs, the LIM domains, which are protein-interacting domains. Using the cDNA of the previously described pollen LIM protein PLIM1 from sunflower as a hybridization probe we have isolated the coding sequence for a related protein from cDNA libraries from various sunflower organs. This protein, WLIM1, is 188 amino acids long and, like the pollen protein PLIM1, contains two LIM domains, separated by a 48 residue spacer region. The two sunflower proteins are structurally related to the animal LIM proteins CRP and MLP. A WLIM1 gene transcript was detected by RT-PCR in all vegetative and reproductive plant organs tested. Polyclonal antibodies raised against the bacterially expressed and affinity-purified protein recognize a polypeptide of ca. 50 kDa in these organs. Immunocytochemical studies detect the protein in many cell types in each of these organs where it is localized either to the cytoplasm, the nucleus, or both. The protein is often associated with plastids and smaller cellular structures or organelles. In late anaphase and early telophase of dividing cells from ovaries, stems and roots it accumulates in the phragmoplast, and may therefore also play a role in cytokinesis.

SF21 was originally described as a pollen- and pistil-expressed protein from sunflower and tobacco. In pistils excised from these species, transcripts were detected in the stigma and in the transmitting tissue where they accumulated in an ovary-oriented increasing concentration gradient. We studied the cellular localization of the SF21 protein during various stages of pistil development as well as in pollen grains of tobacco and sunflower. Here we demonstrate that in young tobacco pistils (from stage 2 onwards) this protein is expressed exclusively in the papillae and secretory cells of the stigma where it is located first in the nucleus and subsequently also in the cytoplasm. Only several stages later (stage 10) does it appear in the transmitting tissue cells of the style where it exhibits a similar, but temporally-delayed, dual nuclear and cytoplasmic localization pattern. SF21 is no longer present in either the stigma or style at the time of pollination, indicating that it is not directly involved in the pollination process. In tobacco pollen grains, SF21 appears just prior to pollen germination and localizes to the apical region of growing pollen tubes, suggesting a possible role in pollen tube growth. This temporal and spatial expression pattern as well as the dual nuclear/cytoplasmic localization suggest that SF21 could be involved in several molecular functions during pistil development and pollen tube growth.