Seed germination characteristics of Phillyrea angustifolia L. and P. latifolia L. (Oleaceae), two Mediterranean shrub species having lignified endocarp

The aim of this study was to determine the germination characteristics of Phillyrea angustifolia L. and P. latifolia L. seeds in order to develop an optimized propagation protocol for Phillyrea species. Seeds of P. angustifolia and P. latifolia were collected from wild plants growing in Cáceres province (CW Spain) and Andalucía (S Spain), respectively. Percentage of water uptake for P. latifolia seeds was calculated. Seeds with and without endocarp were germinated at different constant and alternating temperatures. Seeds without endocarp were soaked in distilled water or gibberellic acid, and then set to germinate. Seeds with endocarp of both species were stratified at 5 ºC for 30 or 90 days and then the endocarp was completely removed from the seeds before they were sowed. Chemical scarification with sulfuric acid and mechanical scarification were tested on P. angustifolia seeds with endocarp. Phillyrea endocarp was permeable to water, since Phillyrea seeds with endocarp imbibed water, but water uptake was faster when the endocarp was removed. Moreover, the encodarp could interfere mechanically in the emergence of the radicle, since seed germination of Phillyrea species was promoted by the complete removal of the lignified endocarp surrounding each seed. Optimal germination temperature for both species was 15 ºC, and lower temperatures produced secondary dormancy. Soaking in distilled water or gibberellic acid did not significantly enhance seed germination. Cold stratification and chemical scarification treatments were detrimental for seed germination. Keywords cold stratification, Phillyrea species, treatments before sowing, seed germination, seed scarification, lignified endocarp.

Mannans and endo-beta-mannanases (MAN) in Brachypodium distachyon: expression profiling and possible role of the BdMAN genes during coleorhiza-limited seed germination

Immunolocalization of mannans in the seeds of Brachypodium distachyon reveals the presence of these polysaccharides in the root embryo and in the coleorhiza in the early stages of germination (12h), decreasing thereafter to the point of being hardly detected at 27h. Concurrently, the activity of endo-β-mannanases (MANs; EC 3.2.1.78) that catalyse the hydrolysis of β-1,4 bonds in mannan polymers, increases as germination progresses. The MAN gene family is represented by six members in the Brachypodium genome, and their expression has been explored in different organs and especially in germinating seeds. Transcripts of BdMAN2, BdMAN4 and BdMAN6 accumulate in embryos, with a maximum at 24–30h, and are detected in the coleorhiza and in the root by in situ hybridization analyses, before root protrusion (germination sensu stricto). BdMAN4 is not only present in the embryo root and coleorhiza, but is abundant in the de-embryonated (endosperm) imbibed seeds, while BdMAN2 and BdMAN6 are faintly expressed in endosperm during post-germination (36–42h). BdMAN4 and BdMAN6 transcripts are detected in the aleurone layer. These data indicate that BdMAN2, BdMAN4 and BdMAN6 are important for germination sensu stricto and that BdMAN4 and BdMAN6 may also influence reserve mobilization. Whether the coleorhiza in monocots and the micropylar endosperm in eudicots have similar functions, is discussed.

Specific interaction of the yeast cis-Golgi syntaxin Sed5p and the coat protein complex II component Sec24p of endoplasmic reticulum-derived transport vesicles

The generation of transport vesicles at the endoplasmic reticulum (ER) depends on cytosolic proteins, which, in the form of subcomplexes (Sec23p/Sec24p; Sec13p/Sec31p) are recruited to the ER membrane by GTP-bound Sar1p and form the coat protein complex II (COPII). Using affinity chromatography and two-hybrid analyses, we found that the essential COPII component Sec24p, but not Sec23p, binds to the cis-Golgi syntaxin Sed5p. Sec24p/Sed5p interaction in vitro was not dependent on the presence of [Sar1p⋅GTP]. The binding of Sec24p to Sed5p is specific; none of the other seven yeast syntaxins bound to this COPII component. Whereas the interaction site of Sec23p is within the N-terminal half of the 926-aa-long Sec24p (amino acid residues 56–549), Sed5p binds to the N- and C-terminal halves of the protein. Destruction by mutagenesis of a potential zinc finger within the N-terminal half of Sec24p led to a nonfunctional protein that was still able to bind Sec23p and Sed5p. Sec24p/Sed5p binding might be relevant for cargo selection during transport-vesicle formation and/or for vesicle targeting to the cis-Golgi.

Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene

With the aim of improving the nutritive value of an important grain legume crop, a chimeric gene specifying seed-specific expression of a sulfur-rich, sunflower seed albumin was stably transformed into narrow-leafed lupin (Lupinus angustifolius L.). Sunflower seed albumin accounted for 5% of extractable seed protein in a line containing a single tandem insertion of the transferred DNA. The transgenic seeds contained less sulfate and more total amino acid sulfur than the nontransgenic parent line. This was associated with a 94% increase in methionine content and a 12% reduction in cysteine content. There was no statistically significant change in other amino acids or in total nitrogen or total sulfur contents of the seeds. In feeding trials with rats, the transgenic seeds gave statistically significant increases in live weight gain, true protein digestibility, biological value, and net protein utilization, compared with wild-type seeds. These findings demonstrate the feasibility of using genetic engineering to improve the nutritive value of grain crops.

Expression of alfalfa mosaic virus coat protein in tobacco mosaic virus (TMV) deficient in the production of its native coat protein supports long-distance movement of a chimeric TMV

Alfalfa mosaic virus (AlMV) coat protein is involved in systemic infection of host plants, and a specific mutation in this gene prevents the virus from moving into the upper uninoculated leaves. The coat protein also is required for different viral functions during early and late infection. To study the role of the coat protein in long-distance movement of AlMV independent of other vital functions during virus infection, we cloned the gene encoding the coat protein of AlMV into a tobacco mosaic virus (TMV)-based vector Av. This vector is deficient in long-distance movement and is limited to locally inoculated leaves because of the lack of native TMV coat protein. Expression of AlMV coat protein, directed by the subgenomic promoter of TMV coat protein in Av, supported systemic infection with the chimeric virus in Nicotiana benthamiana, Nicotiana tabacum MD609, and Spinacia oleracea. The host range of TMV was extended to include spinach as a permissive host. Here we report the alteration of a host range by incorporating genetic determinants from another virus.

Directed seed dispersal by bellbirds in a tropical cloud forest

A fundamental goal of plant population ecology is to understand the consequences for plant fitness of seed dispersal by animals. Theories of seed dispersal and tropical forest regeneration suggest that the advantages of seed dispersal for most plants are escape from seed predation near the parent tree and colonization of vacant sites, the locations of which are unpredictable in space and time. Some plants may gain in fitness as a fortuitous consequence of disperser behavior if certain species of dispersers nonrandomly place seeds in sites predictably favorable for seedling establishment. Such patterns of directed dispersal by vertebrates long have been suggested but never demonstrated for tropical forest trees. Here we report the pattern of seed distribution and 1-year seedling survival generated by five species of birds for a neotropical, shade-tolerant tree. Four of the species dispersed seeds to sites near the parent trees with microhabitat characteristics similar to those at random locations, whereas the fifth species, a bellbird, predictably dispersed seeds under song perches in canopy gaps. The pattern of seedling recruitment was bimodal, with a peak near parent trees and a second peak, corresponding to bellbird song perches, far (>40 m) from parent trees. Seedling survival was higher for seeds dispersed by bellbirds than by the other species, because of a reduction in seedling mortality by fungal pathogens in gaps. Thus, bellbirds play a significant role in seed dispersal by providing directed dispersal to favorable sites and therefore may influence plant recruitment patterns and species diversity in Neotropical forests.

Changes in left ventricular structure and function in patients with white coat hypertension: cross sectional survey

Objectives: To assess the relation between white coat hypertension and alterations of left ventricular structure and function.

The surface coat of procyclic Trypanosoma brucei: Programmed expression and proteolytic cleavage of procyclin in the tsetse fly

Trypanosoma brucei, the protozoan parasite causing sleeping sickness, is transmitted by a tsetse fly vector. When the tsetse takes a blood meal from an infected human, it ingests bloodstream form trypanosomes that quickly differentiate into procyclic forms within the fly's midgut. During this process, the parasite loses the 107 molecules of variant surface glycoprotein that formed its surface coat, and it develops a new coat composed of several million procyclin molecules. Procyclins, the products of a small multigene family, are glycosyl phosphatidylinositol-anchored proteins containing characteristic amino acid repeats at the C terminus [either EP (EP procyclin, a form of procyclin rich in Glu-Pro repeats) or GPEET (GPEET procyclin, a form of procyclin rich in Glu-Pro-Glu-Glu-Thr repeats)]. We have used a sensitive and accurate mass spectrometry method to analyze the appearance of different procyclins during the establishment of midgut infections in tsetse flies. We found that different procyclin gene products are expressed in an orderly manner. Early in the infection (day 3), GPEET2 is the only procyclin detected. By day 7, however, GPEET2 disappears and is replaced by several isoforms of glycosylated EP, but not the unglycosylated isoform EP2. Unexpectedly, we discovered that the N-terminal domains of all procyclins are quantitatively removed by proteolysis in the fly, but not in culture. These findings suggest that one function of the protease-resistant C-terminal domain, containing the amino acid repeats, is to protect the parasite surface from digestive enzymes in the tsetse fly gut.

Cloning and Characterization of TPE4A, a Thiol-Protease Gene Induced during Ovary Senescence and Seed Germination in Pea1

A cDNA clone encoding a thiol-protease (TPE4A) was isolated from senescent ovaries of pea (Pisum sativum) by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of TPE4A has the conserved catalytic amino acids of papain. It is very similar to VSCYSPROA, a thiol-protease induced during seed germination in common vetch. TPE4A mRNA levels increase during the senescence of unpollinated pea ovaries and are totally suppressed by treatment with gibberellic acid. In situ hybridization indicated that TPE4A mRNA distribution in senescent pea ovaries is different from that of previously reported thiol-proteases induced during senescence, suggesting the involvement of different proteases in the mobilization of proteins from senescent pea ovaries. TPE4A is also induced during the germination of pea seeds, indicating that a single protease gene can be induced during two different physiological processes, senescence and germination, both of which require protein mobilization.

Arabidopsis Sec21p and Sec23p Homologs. Probable Coat Proteins of Plant COP-Coated Vesicles1

Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (coat protein)-coated vesicles. Their existence in plant cells has not yet been demonstrated, although the GTP-binding proteins required for coat formation have been identified. We have generated antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p homologs (AtSec21p and AtSec23p, respectively). The former is a constituent of the COPI vesicle coatomer, and the latter is part of the Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower (Brassica oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cytosol and membrane fractions of the cell. The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic proteins. The amounts of the cytosolic antigens relative to the membrane-bound forms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding nonplant coat proteins. Sucrose-density-gradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to be preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes. This could be in agreement with the notion that COPII vesicles are formed at the ER, whereas COPI vesicles can be made by both Golgi and ER membranes. Both AtSec21p and AtSec23p antigens were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems. Therefore, a further purification of the putative plant COP vesicles was undertaken.

Temporal and Spatial Patterns of Accumulation of the Transcript of Myo-Inositol-1-Phosphate Synthase and Phytin-Containing Particles during Seed Development in Rice1

Myo-inositol-1-phosphate (I[1]P) synthase (EC 5.5.1.4) catalyzes the reaction from glucose 6-phosphate to I(1)P, the first step of myo-inositol biosynthesis. Among the metabolites of I(1)P is inositol hexakisphosphate, which forms a mixed salt called phytin or phytate, a storage form of phosphate and cations in seeds. We have isolated a rice (Oryza sativa L.) cDNA clone, pRINO1, that is highly homologous to the I(1)P synthase from yeast and plants. Northern analysis of total RNA showed that the transcript accumulated to high levels in embryos but was undetectable in shoots, roots, and flowers. In situ hybridization of developing seeds showed that the transcript first appeared in the apical region of globular-stage embryos 2 d after anthesis (DAA). Strong signals were detected in the scutellum and aleurone layer after 4 DAA. The level of the transcript in these cells increased until 7 DAA, after which time it gradually decreased. Phytin-containing particles called globoids appeared 4 DAA in the scutellum and aleurone layer, coinciding with the localization of the RINO1 transcript. The temporal and spatial patterns of accumulation of the RINO1 transcript and globoids suggest that I(1)P synthase directs phytin biosynthesis in rice seeds.

Age-Induced Protein Modifications and Increased Proteolysis in Potato Seed-Tubers1

Long-term aging of potato (Solanum tuberosum) seed-tubers resulted in a loss of patatin (40 kD) and a cysteine-proteinase inhibitor, potato multicystatin (PMC), as well as an increase in the activities of 84-, 95-, and 125-kD proteinases. Highly active, additional proteinases (75, 90, and 100 kD) appeared in the oldest tubers. Over 90% of the total proteolytic activity in aged tubers was sensitive to trans-epoxysuccinyl-l-leucylamido (4-guanidino) butane or leupeptin, whereas pepstatin was the most effective inhibitor of proteinases in young tubers. Proteinases in aged tubers were also inhibited by crude extracts or purified PMC from young tubers, suggesting that the loss of PMC was responsible for the age-induced increase in proteinase activity. Nonenzymatic oxidation, glycation, and deamidation of proteins were enhanced by aging. Aged tubers developed “daughter” tubers that contained 3-fold more protein than “mother” tubers, with a polypeptide profile consistent with that of young tubers. Although PMC and patatin were absent from the older mother tubers, both proteins were expressed in the daughter tubers, indicating that aging did not compromise the efficacy of genes encoding PMC and patatin. Unlike the mother tubers, proteinase activity in daughter tubers was undetectable. Our results indicate that tuber aging nonenzymatically modifies proteins, which enhances their susceptibility to breakdown; we also identify a role for PMC in regulating protein turnover in potato tubers.

A Fiberless Seed Mutation in Cotton Is Associated with Lack of Fiber Cell Initiation in Ovule Epidermis and Alterations in Sucrose Synthase Expression and Carbon Partitioning in Developing Seeds1

Fiber cell initiation in the epidermal cells of cotton (Gossypium hirsutum L.) ovules represents a unique example of trichome development in higher plants. Little is known about the molecular and metabolic mechanisms controlling this process. Here we report a comparative analysis of a fiberless seed (fls) mutant (lacking fibers) and a normal (FLS) mutant to better understand the initial cytological events in fiber development and to analyze the metabolic changes that are associated with the loss of a major sink for sucrose during cellulose biosynthesis in the mutant seeds. On the day of anthesis (0 DAA), the mutant ovular epidermal cells lacked the typical bud-like projections that are seen in FLS ovules and are required for commitment to the fiber development pathway. Cell-specific gene expression analyses at 0 DAA showed that sucrose synthase (SuSy) RNA and protein were undetectable in fls ovules but were in abundant, steady-state levels in initiating fiber cells of the FLS ovules. Tissue-level analyses of developing seeds 15 to 35 DAA revealed an altered temporal pattern of SuSy expression in the mutant relative to the normal genotype. Whether the altered programming of SuSy expression is the cause or the result of the mutation is unknown. The developing seeds of the fls mutant have also shown several correlated changes that represent altered carbon partitioning in seed coats and cotyledons as compared with the FLS genotype.

wrinkled1: A Novel, Low-Seed-Oil Mutant of Arabidopsis with a Deficiency in the Seed-Specific Regulation of Carbohydrate Metabolism1

During oil deposition in developing seeds of Arabidopsis, photosynthate is imported in the form of carbohydrates into the embryo and converted to triacylglycerols. To identify genes essential for this process and to investigate the molecular basis for the developmental regulation of oil accumulation, mutants producing wrinkled, incompletely filled seeds were isolated. A novel mutant locus, wrinkled1 (wri1), which maps to the bottom of chromosome 3 and causes an 80% reduction in seed oil content, was identified. Wild-type and homozygous wri1 mutant plantlets or mature plants were indistinguishable. However, developing homozygous wri1 seeds were impaired in the incorporation of sucrose and glucose into triacylglycerols, but incorporated pyruvate and acetate at an increased rate. Because the activities of several glycolytic enzymes, in particular hexokinase and pyrophosphate-dependent phosphofructokinase, are reduced in developing homozygous wri1 seeds, it is suggested that WRI1 is involved in the developmental regulation of carbohydrate metabolism during seed filling.

Yeast Gga Coat Proteins Function with Clathrin in Golgi to Endosome Transport

Gga proteins represent a newly recognized, evolutionarily conserved protein family with homology to the “ear” domain of the clathrin adaptor AP-1 γ subunit. Yeast cells contain two Gga proteins, Gga1p and Gga2p, that have been proposed to act in transport between the trans-Golgi network and endosomes. Here we provide genetic and physical evidence that yeast Gga proteins function in trans-Golgi network clathrin coats. Deletion of Gga2p (gga2Δ), the major Gga protein, accentuates growth and α-factor maturation defects in cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. Cells carrying either gga2Δ or a deletion of the AP-1 β subunit gene (apl2Δ) alone are phenotypically normal, but cells carrying both gga2Δ and apl2Δ are defective in growth, α-factor maturation, and transport of carboxypeptidase S to the vacuole. Disruption of both GGA genes and APL2 results in cells so severely compromised in growth that they form only microcolonies. Gga proteins can bind clathrin in vitro and cofractionate with clathrin-coated vesicles. Our results indicate that yeast Gga proteins play an important role in cargo-selective clathrin-mediated protein traffic from the trans-Golgi network to endosomes.