A cytoskeletal basis for wood formation in angiosperm trees: the involvement of cortical microtubules

Rearrangements of cortical microtubules (CMTs) during the differentiation of axial secondary xylem elements within taproots and shoots of Aesculus hippocastanum L. (horse-chestnut) are described. A correlative approach was employed using indirect immunofluorescence microscopy of α-tubulin in 6- to 10-μm sections and transmission electron microscopy of ultrathin sections. All cell types – fibres, vessel elements and axial parenchyma – derive from fusiform cambial cells which contain randomly oriented CMTs. At the early stages of development, fibres and axial parenchyma cells possess helically arranged CMTs, which increase in number as secondary wall thickening proceeds and simple pits develop. In contrast, incipient vessel elements are distinguished by the marking out of sites of bordered pits; these sites first appear as microtubule-free regions within the reticulum of randomly oriented CMTs that characterises their precursor fusiform cambial cells. Subsequently, the ring of CMTs which develops at the periphery of the microtubule-free region decreases in diameter as the over-arching pit border is formed. Like bordered pits, large-diameter, non-bordered pits (contact pits) which develop between vessel elements and adjacent contact ray cells originate as microtubule-free regions and are also associated with development of a ring of CMTs at the periphery. In the case of contact pits, however, there is no reduction in the diameter of the CMT ring during pit development. Tertiary cell wall thickenings are also a feature of vessel elements and appear to form at sites where bands of laterally associated, transversely oriented CMTs, separated from each other by microtubule-free zones, are found. Later, these bands of CMTs become narrower, and separate into pairs of microtubule bundles located on each side of the developing wall thickening. Development of perforations between vessel elements is also associated with the presence of a ring of CMTs at their periphery. Received: 13 July 1998 / Accepted: 30 November 1998     

Effects of environmental factors on wood formation in larch (Larix sibirica Ldb.) stems

 Effects of temperature and precipitation on xylem cell production by the cambium, radial cell expansion and secondary wall thickening in larch stems have been studied. The observations were carried out over two seasons on ten 50- to 60-year-old trees, growing in central Siberia and chosen according to growth rate (the number of cells in radial rows of each of two of the preceding seasons was equal). The data on the number of cells in differentiation zones and mature xylem along radial rows of tracheids, radial and tangential sizes of tracheids and their lumina were used for calculating cambial activity, the rates and durations of cell development in the zones, and both the thickness and cross-sectional areas of tracheid walls. The mean day air temperature, mean maximum diurnal and mean minimum nocturnal temperatures as well as precipitation have been shown by correlation and regression analyses to affect differentially separate stages of tracheid differentiation. Throughout all the seasons it was temperature that had the main influence on the initial divisions in the xylem, radial cell expansion and biomass accumulation. However, the levels of such an effect on separate stages of cytogenesis were different, especially the influence of nocturnal temperature on xylem cell production by cambium and primary wall growth. The optimum values of temperature and precipitation for cell production by cambium, for radial cell expansion and secondary wall thickening have been calculated. These optimum values of the first and second processes proved to be practically equal, while the last differs considerably in response to temperature. The data are discussed in connection with formation of early and late tracheids. Received: 3 July 1996 / Accepted: 7 February 1997     

Wood formation in poplar: identification, characterization, and seasonal variation of xylem proteins

Proteins that are preferentially produced in developing xylem may play a substantial role in xylogenesis. To reveal the identity of these proteins, comparative two-dimensional polyacrylamide gel electrophoresis was performed on young differentiating xylem, mature xylem, and bark of poplar (Populus trichocarpa Hook. cv. `Trichobel') harvested at different times of the year. The most-abundant xylem proteins were identified by microsequence analysis. For 17 of these proteins a putative function could be assigned based on similarity with previously characterized proteins, and for 15 out of these corresponding expressed sequence tags (ESTs) were found in the poplar EST database. The identified xylem–preferential proteins, defined by comparing the protein patterns from xylem and bark, were all involved in the phenylpropanoid pathway: two caffeoyl-coenzyme A O-methyltransferases (CCoAOMT), one phenylcoumaran benzylic ether reductase (PCBER), one bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), five S-adenosyl-L-methionine synthetases, and one homologue of glycine hydroxymethyltransferase (GHMT). Remarkably, the biological function of the two most-abundant xylem-preferential proteins (PCBER and a GHMT homologue) remains unclear. In addition, several housekeeping enzymes were identified: two enolases, two glutamine synthetases, one 70-kDa heat-shock cognate, one calreticulin, and one α-tubulin. In comparison to the xylem-preferential proteins, the housekeeping proteins were expressed at significant levels in the bark as well. Also, several additional protein spots were detected for CCoAOMT, PCBER, and COMT by immunoblot. Our data show that for the study of xylogenesis, two-dimensional protein gel comparisons combined with systematic protein sequencing may yield information complementary to that from EST sequencing strategies. Received: 28 June 1999 / Accepted: 3 September 1999     

Reaction of pyridoxamine with malondialdehyde: Mechanism of inhibition of formation of advanced lipoxidation end-products

Summary. Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs) are implicated in many age-related chronic diseases and in protein aging. Recent studies suggest that pyridoxamine (PM) is an efficient AGEs/ALEs inhibitor in various biological systems. Because malondialdehyde (MDA) is an important intermediate in the formation of ALEs during lipid peroxidation, the purpose of this study is to determine whether PM can trap MDA directly and thereby prevent ALEs formation. PM reacted readily with MDA under physiological conditions. Within 6 h, a 1-pyridoxamino-propenal adduct derived from reaction of equimolar PM + MDA was detected. A 1-amino-3-iminopropene complex and a dihydropyridine-pyridinium complex were also identified after 7 d incubation. PM also greatly inhibited the lipofuscin-like fluorescence formation induced by MDA reaction with bovine serum albumin (BSA). Our results showed clearly that PM inhibited the formation of ALEs by trapping MDA directly under physiological condition, and provide insight into the mechanism of action of PM in protecting proteins against carbonyl stress.     

Cortical microtubule involvement in bordered pit formation in secondary xylem vessel elements ofAesculus hippocastanum L. (Hippocastanaceae): A correlative study using electron microscopy and indirect immunofluorescence microscopy

Summary A correlative study, using indirect immunofluorescence microscopy (IIF) of anti--tubulin stained sections and transmission electron microscopy (TEM), gave details of the involvement of cortical microtubules (CMTs) in the development of bordered pits in secondary xylem vessel elements ofAesculus hippocastanum L. In addition, aspects of wall cytochemistry were studied during this process using the Thiéry (PATAg) test, immunolocalization with the monoclonal antibodies JIM5 and JIM7, and a range of other cytochemical procedures. IIF showed that the alternately-arranged pits are pre-figured as perforations within a reticulum of randomly-oriented CMTs before any secondary wall thickening is obvious. Each incipient pit border is subsequently delimited by a circle of CMTs whose diameter decreases as deposition of secondary wall takes place around the perforation. These IIF observations are corroborated by a parallel TEM study. During the period of bordered pit formation, the secondary walls of the cell are lignifying. At maturity, however, the pit membrane is unlignified and continues to stain strongly with the monoclonal antibody JIM5, a marker of primary, juvenile wall. The results are discussed in terms of the relationship of the CMT cytoskeleton with development of bordered pits.Abbrevations BSA bovine serum albumin - CMT(s) cortical micro-tubule(s) - EGTA ethylene glycol-bis-(ß-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - IIP indirect immunofluorescence - MAP microtubule-associated protein - mf(s) wall microfibril(s) - MTSB microtubule-stabilising buffer - PATAg periodic acid-thiocarbohydrazide-silver proteinate - PBS Phosphate-buffered saline - PIPES piperazine-N,N-bis-[2-ethylsulphonic acid] - SVS secondary vascular system - TEM transmission electron microscopy     

Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases

 A new system to study programmed cell death in plants is described. Tomato (Lycopersicon esculentum Mill.) suspension cells were induced to undergo programmed cell death by treatment with known inducers of apoptosis in mammalian cells. This chemical-induced cell death was accompanied by the characteristic features of apoptosis in animal cells, such as typical changes in nuclear morphology, the fragmentation of the nucleus and DNA fragmentation. In search of processes involved in plant apoptotic cell death, specific enzyme inhibitors were tested for cell-death-inhibiting activity. Our results showed that proteolysis plays a crucial role in apoptosis in plants. Furthermore, caspase-specific peptide inhibitors were found to be potent inhibitors of the chemical-induced cell death in tomato cells, indicating that, as in animal systems, caspase-like proteases are involved in the apoptotic cell death pathway in plants. Received: 5 August 1999 / Accepted: 14 March 2000     

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures

Maize (Zea mays L.) cell cultures incorporated radioactivity from [¹⁴C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the CoA pool had lost its ¹⁴C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1–3 d) cultures, polysaccharide-bound [¹⁴C]feruloyl- and [¹⁴C]diferuloyl residues were both detectable within 1 min of [¹⁴C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least the first 2.3 h after [¹⁴C]cinnamate feeding, polysaccharide-bound [¹⁴C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter, and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H₂O₂ (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [¹⁴C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H₂O₂ induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H₂O₂-limited. In both 2- and 4-d-old cultures, polysaccharide-bound ¹⁴C-trimers and larger coupling products exceeded [¹⁴C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed. Received: 19 January 2000 / Accepted: 13 April 2000     

Polyamine contents in current foods: a basis for polyamine reduced diet and a study of its long term observance and tolerance in prostate carcinoma patients

Summary. Our aim was to develop a liquid chromatography and electrospray ionization tandem mass spectrometry (LCMS²) method to measure free amino acid (FAA) and dipeptide (DP) concentrations in biological fluids. We synthesized chloroformate derivatives of FAA and DP, identified the major precursor ions and used LCMS² to obtain the most intense product ions. Using serial dilutions of unlabeled and labeled standards ([²H₃]-L-Dopa, homoarginine, homophenylalanine, [¹⁵N]-Glutamine and [²H₃]-methionine), we observed linear relationships in MS response that we used to calculate the amounts of FAA and DP in biological samples. This method is sensitive with a limit of detection (LOD) for most of the FAAs and DPs tested in the 0.05–1 pmol range and is linear over 3–5 orders of magnitude when many metabolites were measured simultaneously. Reproducibility and between run or daily variations were <10% for most FAAs and DPs. We applied this method to human samples and quantitatively measured 21 FAAs and 2 DPs in 200 μl CSF, 31 FAAs and 6 DPs in 100 μl plasma, and 23 FAAs and 5 DPs in 200 μl urine. These data demonstrate the potential for using LCMS² to discover changes in FAA and DP metabolic pathways that occur during disease pathogenesis.     

Mechanisms of primordium formation during adventitious root development from walnut cotyledon explants

 In walnut (Juglans regia L.), an otherwise difficult-to-root species, explants of cotyledons have been shown to generate complete roots in the absence of exogenous growth regulators. In the present study, this process of root formation was shown to follow a pattern of adventitious, rather than primary or lateral, ontogeny: (i) the arrangement of vascular bundles in the region of root formation was of the petiole type; (ii) a typical root primordium was formed at the side of the procambium within a meristematic ring of actively dividing cells located around each vascular bundle; (iii) the developing root apical meristem was connected in a lateral way with the vascular bundle of the petiole. This adventitious root formation occurred in three main stages of cell division, primordium formation and organization of apical meristem. These stages were characterized by expression of LATERAL ROOT PRIMORDIUM-1 and CHALCONE SYNTHASE genes, which were found to be sequentially expressed during the formation of the primordium. Activation of genes related to root cell differentiation started at the early stage of primordium formation prior to organization of the root apical meristem. The systematic development of adventitious root primordia at a precise site gave indications on the positional and biochemical cues that are necessary for adventitious root formation. Received: 30 July 1999 / Accepted: 16 February 2000     

Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells

 Active auxin transport in plant cells is catalyzed by two carriers working in opposite directions at the plasma membrane, the influx and efflux carriers. A role for the efflux carrier in polar auxin transport (PAT) in plants has been shown from studies using phytotropins. Phytotropins have been invaluable in demonstrating that PAT is essential to ensure polarized and coordinated growth and to provide plants with the capacity to respond to environmental stimuli. However, the function of the influx carrier at the whole-plant level is unknown. Our work aims to identify new auxin-transport inhibitors which could be employed to investigate its function. Thirty-five aryl and aryloxyalkylcarboxylic acids were assayed for their ability to perturb the accumulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (1-NAA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. As 2,4-D and 1-NAA are preferentially transported by the influx and efflux carriers, respectively, accumulation experiments utilizing synthetic auxins provide independant information on the activities of both carriers. The majority (60%) of compounds half-inhibited the carrier-mediated influx of [¹⁴C]2,4-D at concentrations of less than 10 μM. Most failed to interfere with [³H]NAA efflux, at least in the short term. Even though they increasingly perturbed auxin efflux when given a prolonged treatment, several compounds were much better at discriminating between influx and efflux carrier activities than naphthalene-2-acetic acid which is commonly employed to investigate influx-carrier properties. Structure-activity relationships and factors influencing ligand specificity with regard to auxin carriers are discussed. Received: 28 June 1999 / Accepted: 28 August 1999     

Phenylcoumaran benzylic ether reductase, a prominent poplar xylem protein, is strongly associated with phenylpropanoid biosynthesis in lignifying cells

 It has previously been shown (D.R. Gang et al., 1999, J Biol Chem 274: 7516–7527) that the most abundant protein in the secondary xylem of poplar (Populus trichocarpa cv. `Trichobel') is a phenylcoumaran benzylic ether reductase (PCBER), an enzyme involved in lignan synthesis. Here, the distribution and abundance of PCBER in poplar was studied at both the RNA and protein level. The cellular expression pattern was determined by immunolocalization of greenhouse-grown plants as well as of a field-grown poplar. Compared to other poplar tissues, PCBER is preferentially produced in the secondary xylem of stems and roots and is associated with the active growth period. The protein is present in all cells of the young differentiating xylem, corresponding to the zone of active phenylpropanoid metabolism and lignification. In addition, PCBER is located in young differentiating phloem fibers, in xylem ray parenchyma, and in xylem parenchyma cells at the growth-ring border. Essentially the same expression pattern was observed in poplars grown in greenhouses and in the field. The synthesis of PCBER in phenylpropanoid-synthesizing tissues was confirmed in a bending experiment. Induction of PCBER was observed in the pith of mechanically bent poplar stems, where phenylpropanoid metabolism is induced. These results indicate that the products of PCBER activity are synthesized mainly in lignifying tissues, suggesting a role in wood development. Received: 28 September 1999 / Accepted: 15 March 2000     

A nonphotochemical-quenching-deficient mutant of Arabidopsis thaliana possessing normal pigment composition and xanthophyll-cycle activity

Higher-plant chloroplasts alter the distribution of absorbed radiant energy between photosynthesis and heat formation in response to changing illumination level or environmental stress. Fluorescence imaging was used to screen 62 yellow-green T-DNA insertion mutant lines of Arabidopsis thaliana (L.) Heynh. for reduced photoprotective nonphotochemical quenching (NPQ) capacity. Pulse-modulation fluorometry was employed to characterize one line (denoted Lsr1⁻) that exhibited an approximately 50% reduction in NPQ compared to the wild type (WT). The loss in NPQ capacity was associated with the ΔpH-dependent phase of quenching (qE). Under the growth conditions employed, pigment composition and levels of the six photosystem-II light-harvesting chlorophyll a/b proteins were identical in mutant and WT. Changes in the in-vivo levels of the xanthophyll pigments violaxanthin, antheraxanthin, and zeaxanthin in excess light were the same for mutant and WT. However, use of the violaxanthin de-epoxidase inhibitor dithiothreitol indicated that a zeaxanthin-dependent component of NPQ was specifically reduced in the mutant. The mutant exhibited diminished suppression of minimum fluorescence yield (F _o ) in intense light suggesting an altered threshold in the mechanism of response to light stress in the mutant. The NPQ-deficient phenotype was meiotically transmissible as a semidominant trait and mapped near marker T27K12 on chromosome 1. The results suggest that the mutant is defective in sensing the transthylakoid ΔpH that reports exposure to excessive illumination. Received: 26 May 1999 / Accepted: 17 June 1999     

Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.)

The temporal and spatial patterns of storage-globulin mobilization were immunohistochemically pursued in the embryonic axis and cotyledons of vetch seed (Vicia sativa L.) during germination and early seedling growth. Embryonic axes as well as cotyledons of mature seeds contain protein bodies with stored globulins. Prevascular strands of axes and cotyledons, the radicle and epidermal layers of axis organs were nearly exclusively stained by vicilin antibodies whereas the cotyledonous storage mesophyll gave similar staining for vicilin and legumin. Globulin breakdown started locally where growth and differentiation commenced in the axis. There, vicilin mobilization preceded legumin mobilization. Thus vicilin represents the initial source of amino acids for early growth and differentiation processes in vetch. Legumin presumably only serves as a bulk amino acid source for subsequent seedling growth during postgerminative globulin degradation. During the first 2–3 d after the start of imbibition the axis was depleted of globulins whereas no decrease in immunostainability was detected in the cotyledons except in their vascular strands where immunostainability was almost completely lost at this time. Continuous vascular strands were established at the third day when globulin breakdown was finished in the axis but had just started in the cotyledon mesophyll. Protein mobilization proceeded in a small zone from the epidermis towards the vascular strands in the center of the cotyledons. In this zone the storage cells, which initially appeared densely packed with starch grains and protein bodies, concomitantly transformed into cells with a large central vacuole and only a thin cytoplasmic layer attached to the cell wall. These results agree well with the hypothesis that during the first 2 d after imbibition the axis is autonomous in amino acid provision. After the endogenous reserves of the axis are depleted and the conductive tissue has differentiated, globulins are mobilized in the cotyledons, suggesting that then the amino acid supply is taken over by the cotyledons. For comparison with other degradation patterns we used garden bean (Phaseolus vulgaris L) and rape (Brassica napus L.) as reference plants. Received: 3 August 1999 / Accepted: 11 December 1999     

A novel reverse-genetic approach (SIMF) identifies Mutator insertions in new Myb genes

We have developed a new strategy designated SIMF (Systematic Insertional Mutagenesis of Families), to identify DNA insertions in many members of a gene family simultaneously. This method requires only a short amino acid sequence conserved in all members of the family to make a degenerate oligonucleotide, and a sequence from the end of the DNA insertion. The SIMF strategy was successfully applied to the large maize R2R3 Myb family of regulatory genes, and Mutator insertions in several novel Myb genes were identified. Application of this technique to identify insertions in other large gene families could significantly decrease the effort involved in screening at the same time for insertions in all members of groups of genes that share a limited sequence identity. Received: 1 June 2000 / Accepted: 26 June 2000     

Control of gravimorphogenesis by auxin: accumulation pattern of CS-IAA1 mRNA in cucumber seedlings grown in space and on the ground

Cucumber (Cucumis sativus L.) seedlings grown in microgravity developed a peg on each side of the transition zone between hypocotyl and root, whereas seedlings grown in a horizontal position on the ground developed a peg on the concave side of the gravitropically bending transition zone. The morphological features of the space-grown seedlings were similar to those of seedlings grown in a vertical position on the ground with their radicles pointing down: both became two-pegged seedlings. Morphogenesis of cucumber seedlings is thus inhibited by gravity. Analysis by in-situ hybridization of an auxin-inducible gene, CS-IAA1, showed that its mRNA accumulated to a much greater extent on the lower side of the transition zone in the horizontally placed seedlings on the ground just prior to and during the initiation period of peg formation. On the other hand, when seedlings were grown in microgravity or in a vertical position on the ground, accumulation of CS-IAA1 mRNA occurred all around the transition zone. Accumulation of CS-IAA1 mRNA in horizontally grown seedlings appreciably decreased on the upper side of the transition zone and increased on the lower side upon gravistimulation, compared with the two-pegged seedlings. Application of IAA to seedlings in a horizontal position caused the development of a peg on each side of the transition zone, or a collar-like protuberance, depending on the concentration used. These results suggest that upon gravistimulation the auxin concentration on the upper side of the horizontally placed transition zone is reduced to a level below the threshold value necessary for peg formation. Space-grown seedlings of cucumber might develop two pegs symmetrically because the auxin level in the entire transition zone is maintained above the threshold. This spaceflight experiment verified for the first time that auxin does not redistribute in microgravity. Received: 10 February 2000 / Accepted: 15 March 2000     

Expression of cucumber lipid-body lipoxygenase in transgenic tobacco: lipid-body lipoxygenase is correctly targeted to seed lipid bodies

 A particular isoform of lipoxygenase (LOX, EC 1.13.11.12) localized on lipid bodies has been shown by earlier investigations to play a role during seed germination in initiating the mobilization of triacylglycerols. On lipid bodies of germinating cucumber (Cucumis sativus L.) seedlings, the modification of linoleoyl moieties by this LOX precedes the hydrolysis of the ester bonds. We analyzed the expression and intracellular location of this particular LOX form in leaves and seeds of tobacco (Nicotiana tabacum L.) transformed with one construct coding for cucumber lipid-body LOX and one construct coding for cucumber LOX fused with a hemagglutinin epitope. In both tissues, the amount of lipid-body LOX was clearly detectable. Biochemical analysis revealed that in mature seeds the foreign LOX was targeted to lipid bodies, and the preferred location of the LOX on lipid bodies was verified by immunofluorescence microscopy. Cells of the endosperm and of the embryo exhibited fluorescence based on the immunodecoration of LOX protein whereas very weak fluorescent label was visible in seeds of untransformed control plants. Further cytochemical analysis of transformed plants showed that the LOX protein accumulated in the cytoplasm when green leaves lacking lipid bodies were analyzed. Increased LOX activity was shown in young leaves of transformed plants by an increase in the amounts of endogenous (2E)-hexenal and jasmonic acid. Received: 9 August 1999 / Accepted: 28 September 1999     

A DEFICIENS homologue is down-regulated during apomictic initiation in ovules of Hieracium

 Hieracium is a member of the Asteraceae family, and contains sexual species in addition to apomictic species that reproduce by apospory and produce seed without fertilization. A homologue of the floral organ-identity gene DEFICIENS (DEF) was isolated from an apomictic line of Hieracium piloselloides (Vill.) following differential display between mature ovules and those initiating autonomous embryogenesis. The gene termed HPDEF has 93% amino acid identity with GDEF2, a DEF homologue isolated from Gerbera hybrida (D. Yu et al., 1999, Plant J. 17: 51–62), another member of the Asteraceae. In-situ analysis showed that early in floral development HPDEF is expressed in stamen and petal primordia, indicating expected B-function activity, according to the ABC model of floral organ identity (J. L. Bowman et al., 1991, Development 112: 1–20; E. S. Coen and E. M. Meyerowitz, 1991, Nature 353: 31–37). However, HPDEF expression was also observed in ovule primordia and expression continued in developing ovules until anthesis, indicating that this gene may have a role in ovule development. Expression of HPDEF was not detected in megaspore mother cells, or in sexual or aposporous embryo sacs. In sexual Hieracium, HPDEF was uniformly expressed throughout the ovule integument until anthesis. In most ovules of the apomict, however, HPDEF expression was transiently down-regulated in a specific zone in the chalazal region where cells initiating aposporous embryo sac formation differentiate. Uniform low-level HPDEF expression was subsequently observed prior to anthesis in ovules from sexual and apomictic plants. HPDEF may be down-regulated as a consequence of apomictic initiation and/or its down-regulation may facilitate progression of apomictic events. Received: 15 September 1999 / Accepted: 12 October 1999