Effect of Low Temperature on Changes in AGP Distribution during Development of Bellis perennis Ovules and Anthers.

Arabinogalactan proteins (AGPs) are a class of heavily glycosylated proteins occurring as a structural element of the cell wall-plasma membrane continuum. The features of AGPs described earlier suggest that the proteins may be implicated in plant adaptation to stress conditions in important developmental phases during the plant reproduction process. In this paper, the microscopic and immunocytochemical studies conducted using specific antibodies (JIM13, JIM15, MAC207) recognizing the carbohydrate chains of AGPs showed significant changes in the AGP distribution in female and male reproductive structures during the first stages of Bellis perennis development. In typical conditions, AGPs are characterized by a specific persistent spatio-temporal pattern of distribution. AGP epitopes are visible in the cell walls of somatic cells and in the megasporocyte walls, megaspores, and embryo sac at every stage of formation. During development in stress conditions, the AGP localization is altered, and AGPs entirely disappear in the embryo sac wall. In the case of male development, AGPs are present in the tapetum, microsporocytes, and microspores in normal conditions. In response to development at lower temperature, AGPs are localized in the common wall of microspores and in mature pollen grains. Additionally, they are accumulated in remnants of tapetum cells.

Seed germination response to high temperature and water stress in three invasive Asteraceae weeds from Xishuangbanna, SW China.

Crassocephalum crepidioides, Conyza canadensis, and Ageratum conyzoides are alien annuals naturalized in China, which produce a large number of viable seeds every year. They widely grow in Xishuangbanna, becoming troublesome weeds that compete with crops for water and nutrients. As seed germination is among the most important life-stages which contribute to plant distribution and invasiveness, its adaptation to temperature and water stress were investigated in these three species. Results showed that: (1) These three species have wide temperature ranges to allow seed germination, i.e., high germination and seedling percentages were achieved between 15°C and 30°C, but germination was seriously inhibited at 35°C; only A. conyzoides demonstrated relative preference for warmer temperatures with approximately 25% germination and seedling percentage at 35°C; (2) light was a vital germination prerequisite for C. crepidioides and A. conyzoides, whereas most C. canadensis seeds germinated in full darkness; (3) Although all three species have good adaptation to bare ground habitat characterized by high temperatures and water stress, including their tolerance to soil surface temperatures of 70°C in air-dried seeds, A. conyzoides seeds exhibited higher tolerance to both continuous and daily periodic high-temperature treatment at 40°C, and to water restriction (e.g., ca. 65% seeds germinated to -0.8 MPa created by NaCl), which is consistent with their field behavior in Xishuangbanna. This study suggests that seed high-temperature tolerance contributes to the weed attributes of these three species, and that adaptation to local micro-habitats is a critical determinant for invasiveness of an alien plant.

Genetic analyses of the inheritance and expressivity of autonomous endosperm formation in Hieracium with different modes of embryo sac and seed formation.

Background and Aims: Apomixis, or asexual seed formation, in polyploid Hieracium subgenus Pilosella species results in clonal progeny with a maternal genotype. An aposporous embryo sac forms mitotically from a somatic cell, without prior meiosis, while embryo and endosperm formation is fertilization independent (autonomous). The latter two developmental components are tightly linked in Hieracium . Recently, two plants, AutE196 and AutE24, were identified from two different crosses. Both form embryo sacs via the sexual route by undergoing meiosis, and embryo development requires fertilization; however, 18 % of embryo sacs can undergo autonomous endosperm (AutE) formation. This study investigated the qualitative and quantitative inheritance of the AutE trait and factors influencing phenotype expressivity. An additional focus was to identify the linkage group bearing the AutE locus in AutE196. Methods: Crosses and cytology were used to examine the inheritance of AutE from AutE24 and AutE196, and to reintroduce apomictic components into AutE plants, thereby changing the ploidy of developing embryo sacs and increasing the dosage of AutE loci. Markers from a Hieracium apomict linkage map were examined within a backcrossed AutE196 mapping population to identify the linkage group containing the AutE196 locus. Key Results: Qualitative autonomous endosperm in the AutE24 line was conferred by a single dominant locus, and the trait was transmitted through male and female gametes in AutE196 and AutE24. Expressivity of the trait did not significantly increase when AutE loci from AutE196 and AutE24 were both present in the progeny, within embryo sacs formed via apospory, or sexually derived embryo sacs with increased ploidy. It remains unclear if these are identical loci. Conclusions: The qualitative trait of autonomous endosperm formation is conferred by single dominant loci in AutE196 and AutE24. High expressivity of autonomous endosperm formation observed in apomicts requires additional genetic factors. Potential candidates may be signals arising from fertilization-independent embryo formation.

[Studies on sexual organs and embryological development morphology of Pterocypsela formosana].

In this study, the embryological characters of Pterocypsela formosana (Asteraceae) were investigated with the traditional paraffin section methods. The anther has 4 sporangiates, the anther wall development follows the dicotyledonous type and comprises of an epidermis, endothelium, a middle layer and a single-layered tapetum, the tapetum belongs to glandular type. Meiosis of the microspore mother cells is of the simultaneous type, for the formation of mostly tetrahedral tetrad, the mature pollen grains are 2 celled. The ovary is bicarpellate, unilocular, one ovule and basal placenta, the ovule is unitegmic, tenuinucellate, inverted campylotropous and with developed endothelium, archesporial cell of megaspore differentiates immediately below the nucellar epidermis and functions as megasporocyte after development and belongs to tenuinucellate ovule type. The megasporocyte undergoes meitotic to form a liner tetrad, only one chalazal megaspore becomes the functional megaspore which forms female gametophyte including 7-celled and 8-nucleated after three successive mitosis, the female gametophyte is of the Polygonum type. Two polar nuclei melt into a secondary nuclei before fertilization, the chalazal antipodal cells are ephemeral and degenerate shortly after forming. Fertilization is porogamous and belongs to premitotic type of syngamy. The division of the primary endosperm nucleus is earlier than the zygote, the endosperm is of the nuclear type with the presence of haustoria, and the embryogeny belongs to asterad type chicory variant. The developed suspensor on early stage has important significance to the embryo development.

The embryo rescue derived intergeneric hybrid between chrysanthemum and Ajania przewalskii shows enhanced cold tolerance.

Five intergeneric hybrids between the chrysanthemum cultivar 'Zhongshanjingui' (as female) and Ajania przewalskii (as male) were obtained with the help of embryo culture. While 'Zhongshanjingui' bears a standard anemone type flower and A. przewalskii a non-anemone type one, the inflorescence type of the hybrids varied. The diameter of the hybrids' flowers was intermediate between those of the parents. The chromosome number of the hybrids was 2n = 45, of which GISH analysis was able to establish that 27 were inherited from 'Zhongshanjingui' and the other 18 from A. przewalskii. A combination of various assays was used to show that the cold tolerance of the hybrids was equivalent to that of the highly tolerant A. przewalskii parent. Enhanced cold tolerance was correlated with an increase in free proline and a decrease in malondialdehyde content.

Plant tissue cultures.

Plant tissue cultures are an efficient system to study cell wall biosynthesis in living cells in vivo. Tissue cultures also provide cells and culture medium where enzymes and cell wall polymers can easily be separated for further studies. Tissue cultures with tracheary element differentiation or extracellular lignin formation have provided useful information related to several aspects of xylem and lignin formation. In this chapter, methods for nutrient medium preparation, callus culture initiation, and its maintenance, as well as those for protoplast isolation and viability observation, are described. As a case study, we describe the establishment of a xylogenic culture of Zinnia elegans mesophyll cells.

Isolation and structure determination of acetylated triterpenoid saponins from the seeds of Centratherum anthelminticum.

Phytochemical investigation of Centratherum anthelminticum seeds resulted in the isolation of two novel glycosides. The pentacyclic triterpenoid saponins were shown to contain hederagenin and sugar residues forming two glycosyl chains. The structural analysis of its acetylated derivative was determined using a combination of homo- (1D 1H NMR, 13C NMR and 13C NMR-DEPT) and heteronuclear 2D NMR techniques (1H-1H-COSY, total correlated spectroscopy (TOCSY), heteronuclear multiple quantum coherence (HMQC)), heteronuclear multiple bond correlation (HMBC) and chemical methods. The structure of the saponins were established to be 3-O-[beta-D-glucopyranosyl-(1 --> 2)-alpha-L-rhamnopyranosyl-(1 --> 2)-alpha-L-arabinopyranosyl]-28-O-[beta-D-xylopyranosyl-(1 --> 4)-alpha-L-rhamnopyranosyl-(1 --> 3)-beta-D-glucopyranosyl]-23-hydroxyolean-12-en-28-oic acid and 3-O-[beta-D-glucopyranosyl-(1 --> 2)-alpha-L-rhamnopyranosyl-(1 --> 2)-alpha-L-arabinopyranosyl]-28-O-[beta-D-glucoyranosyl-(1 --> 3)-beta-D-glucopyranosyl]-23-hydroxyolean-12-en-28-oic acid. Different extracts and compounds were also tested for antifilarial and antimicrobial activities. Methanolic, acetone and aqueous extracts of seeds exhibited some pronounced pharmacological activity under study.

Shifts in leaf vein density through accelerated vein formation in C4 Flaveria (Asteraceae).

BACKGROUND AND AIMS: Leaf venation in many C(4) species is characterized by high vein density, essential in facilitating rapid intercellular diffusion of C(4) photosynthetic metabolites between different tissues (mesophyll, bundle sheath). Greater vein density has been hypothesized to be an early step in C(4) photosynthesis evolution. Development of C(4) vein patterning is thought to occur from either accelerated or prolonged procambium formation, relative to ground tissue development. METHODS: Cleared and sectioned tissues of phylogenetically basal C(3) Flaveria robusta and more derived C(4) Flaveria bidentis were compared for vein pattern in mature leaves and vein pattern formation in developing leaves. KEY RESULTS: In mature leaves, major vein density did not differ between C(3) and C(4) Flaveria species, whereas minor veins were denser in C(4) species than in C(3) species. The developmental study showed that both major and minor vein patterning in leaves of C(3) and C(4) species were initiated at comparable stages (based on leaf length). An additional vein order in the C(4) species was observed during initiation of the higher order minor veins compared with the C(3) species. In the two species, expansion of bundle sheath and mesophyll cells occurred after vein pattern was complete and xylem differentiation was continuous in minor veins. In addition, mesophyll cells ceased dividing sooner and enlarged less in C(4) species than in C(3) species. CONCLUSIONS: Leaf vein pattern characteristic to C(4) Flaveria was achieved primarily through accelerated and earlier offset of higher order vein formation, rather than other modifications in the timing of vein pattern formation, as compared with C(3) species. Earlier cessation of mesophyll cell division and reduced expansion also contributed to greater vein density in the C(4) species. The relatively late expansion of bundle sheath and mesophyll cells shows that vein patterning precedes ground tissue development in C(4) species.

A TCP domain transcription factor controls flower type specification along the radial axis of the Gerbera (Asteraceae) inflorescence.

Several key processes in plant development are regulated by TCP transcription factors. CYCLOIDEA-like (CYC-like) TCP domain proteins have been shown to control flower symmetry in distantly related plant lineages. Gerbera hybrida, a member of one of the largest clades of angiosperms, the sunflower family (Asteraceae), is an interesting model for developmental studies because its elaborate inflorescence comprises different types of flowers that have specialized structures and functions. The morphological differentiation of flower types involves gradual changes in flower size and symmetry that follow the radial organization of the densely packed inflorescence. Differences in the degree of petal fusion further define the distinct shapes of the Gerbera flower types. To study the role of TCP transcription factors during specification of this complex inflorescence organization, we characterized the CYC-like homolog GhCYC2 from Gerbera. The expression of GhCYC2 follows a gradient along the radial axis of the inflorescence. GhCYC2 is expressed in the marginal, bilaterally symmetrical ray flowers but not in the centermost disk flowers, which are nearly radially symmetrical and have significantly less fused petals. Overexpression of GhCYC2 causes disk flowers to obtain morphologies similar to ray flowers. Both expression patterns and transgenic phenotypes suggest that GhCYC2 is involved in differentiation among Gerbera flower types, providing the first molecular evidence that CYC-like TCP factors take part in defining the complex inflorescence structure of the Asteraceae, a major determinant of the family's evolutionary success.

Unusual microtubular cytoskeleton of apomictic embryo sac of Chondrilla juncea L.

The mature apomictic embryo sac of Chondrilla juncea is highly vacuolated and demonstrates a polarization similar to that of the amphimictic gametophyte. The microtubule cytoskeleton of this embryo sac is uncharacteristic and relatively weak. The microtubules are positioned along cell walls and resemble cortical microtubules of somatic cells. They do not form the parallel, brushlike structures observed around the filiform apparatus of synergids in the amphimictic embryo sac. In the apomictic embryo sac, the microtubules of both the egg cell and the central cell develop a cortical-like structure, which is entirely different from the radial arrangement observed around the nuclei in the amphimictic embryo sac.

Somatic embryogenesis from leaf explants of Australian fan flower, Scaevola aemula R. Br.

Somatic embryogenesis from leaf explants of Scaevola aemula R. Br. was achieved. Somatic embryos were induced from explants cultured on MS medium supplemented with 0.2 mg/ 2,4-dichlorophenoxyacetic acid and 0.2-0.5 mg/l 6-benzylaminopurine (BAP). Various developmental stages of somatic embryos were found on this medium-from globular embryos to germinated embryos. The transfer of globular embryos to MS medium containing 0.5 mg/l BAP resulted in a high frequency of shoot regeneration. Leaf explants cultured on MS medium containing different combinations of BAP and alpha-naphthaleneacetic acid formed adventitious shoots and roots. Histological examination confirmed the process of somatic embryogenesis. Induction of somatic embryogenesis in Scaevola provides a system for studying embryogenesis in Australian native plants and will facilitate the improvement of these plants using genetic transformation techniques.

Three major somatic embryogenesis related proteins in Cichorium identified as PR proteins.

In Cichorium hybrid clone '474' (C. intybus L., var. sativum x C. endivia L., var. latifolia), the direct somatic embryogenesis process in leaf tissues is accompanied by an overall increase in the amount of proteins secreted into the culture medium. Amongst these, three major protein bands of 38 kDa, 32 kDa and 25 kDa were found in the conditioned media. These extracellular protein bands accumulated in the medium of the embryogenic Cichorium hybrid up to 8-fold compared with those in the medium of a nonembryogenic variety. 32 and 25 kDa proteins were purified from the medium and their identities were determined as already described for 38 kDa beta-1,3-glucanases. To investigate their possible function in somatic embryogenesis, peptide sequences, serological relationships or biochemical properties revealed that there were at least two acidic chitinases of 32 kDa and one glycosylated osmotin-like protein of 25 kDa in the embryogenic culture medium. Comparing the amounts of the 38 kDa glucanases, the 32 kDa chitinases, and the 25 kDa osmotin-like protein present in the conditioned media of the embryogenic '474' hybrid and of a non-embryogenic variety, a 2-8-fold higher accumulation of these proteins was observed in the embryogenic hybrid culture medium. This may suggest that part of the accumulation of these three pathogenesis-related (PR) proteins could be correlated with the somatic embryogenesis process. Their possible involvement in this developmental process is discussed.

Cloning of beta-1,3-glucanases expressed during Cichorium somatic embryogenesis.

Three different beta-1,3-glucanase cDNA fragments, CG1, CG2 and CG3, were obtained by RT-PCR from RNA isolated from Cichorium hybrid '474' leaf fragments cultured for 11 days under somatic embryogenesis-inducing conditions. When expressed in Escherichia coli the proteins encoded by the three cDNAs were recognized by antibodies raised against 38 kDa extracellular beta-1,3-glucanases studied previously (Helleboid et al., Planta 205 (1998) 56-63). The CG2 and CG3 cDNAs may represent expressed alleles of one gene because their sequences showed a very high identity (98.5%) and are only 70% identical with CG1. Southern blot analysis revealed the presence of 3-4 genes coding for beta-1,3-glucanases in the Cichorium genome. Expression analysis of the genes corresponding to the three clones analysed by semi-quantitative RT-PCR indicated that CG1 mRNAs were only detectable in Cichorium hybrid '474' leaf fragments from day 3 of somatic embryogenesis induction, whereas CG2-CG3 mRNAs were already present in non-induced leaf tissue of both the embryogenic hybrid '474' and a non-embryogenic genotype. The level of CG1 mRNAs was particularly high when embryogenic cells were dividing to produce embryos, and when the amount of callose deposited in cell walls surrounding embryogenic cells and young embryos decreased. These results indicate that expression of the CG1 gene is correlated to the somatic embryogenesis process and that it encodes a 38 kDa beta-1,3-glucanase protein that may be involved in the degradation of callose localized around embryogenic cells and young embryos. A full-length CG1 cDNA clone was obtained using 3' and 5' RACE-PCR, and its sequence revealed that it encodes a beta-1,3-glucanase that is equally homologous to both class III and class IV plant beta-1,3-glucanases.

Direct somatic embryogenesis and plantlet regeneration from leaf explants of niger, Guizotia abyssinica (L.f.) Cass.

Somatic embryogenesis was induced in niger (Guizotia abyssinica (L.f.) Cass.) using a simple one step method. Leaf explants were cultured on MS medium with 2,4-D and BAP/Kn individually and in combination. Somatic embryogenesis occurred directly without an intervening callus proliferation phase from subepidermal regions of leaf explants on MS medium supplemented with 2,4-D (2.0 and 5.0 microM) plus Kn/BAP (1.0 and 2.0 microM). Regenerated somatic embryos were successfully grown into whole plants.