Carpellary outgrowth development in the endocarp of grapefruit,Citrus paradisi (Rutaceae)

The development and function of the carpellary outgrowth was investigated in grapefruit. Carpellary outgrowths were initiated 3 d postanthesis. By 7 d postanthesis, enlarged globose cells developed that were subtended by a parenchymatous stalk. Numerous Golgi, Golgi-derived vesicles, endoplasmic reticulum, mitochondria, and thickened cell walls present in globose cells from 7 d to 3 mo postanthesis suggest that the carpellary outgrowth was secretory in nature. Lectin-gold complex binding indicated that the secreted product contained galactose and arabinose. Cessation of secretory activity, marked by protoplast degeneration, occurred between 3 and 5 mo postanthesis.     

Platelet-derived growth factor A (pdgf-a) expression during zebrafish embryonic development

Zebrafish pdgf-a gene was cloned and its expression pattern studied during zebrafish embryogenesis. Zebrafish pdgf-a mRNA was present at high levels in fertilized eggs as well as in all embryonic cells up to the end of gastrulation. Spatially restricted expression started after the onset of segmentation and was mainly localized in the developing pharyngeal arches. Transient expression was also detected in Kupffer's vesicle, a teleost-specific structure, and in lateral trunk and tail regions surrounding the neural keel, as well as areas of the developing pronephros.     

Secondary metabolism as an expression of microbial growth and development

A case is made out for regarding secondary metabolism as part of normal cell growth, related to its interactions with the environment. Secondary metabolism is widespread, especially in fungi and actinomycetes, and is not to be regarded as confined to the production of antibiotics and other special substances. It is part of the normal maturation process. Examples are given of the influence of secondary metabolism in ecological systems. It is also shown that cell productivity can be related to age structure. Secondary metabolism is thus linked with growth, although in many cases this may not be obvious in laboratory work. Initiation of production will arise from the system which regulates growth and differentiation. These processes are little understood at present, but it is clear that the factors involved differ in different instances and that they involve a very great variety of biochemical and physiological processes.     

Somatic hybridization in Citrus: navel orange (C. sinensis Osb.) and grapefruit (C. paradisi Macf.)

Summary Protoplasts of navel orange, isolated from embryogenic nucellar cell suspension culture, were fused with protoplasts of grapefruit isolated from leaf tissue. The fusion products were cultured in the hormone-free medium containing 0.6 M sucrose. Under the culture conditions, somatic embryogenesis of navel orange protoplasts was suppressed, while cell division of grapefruit mesophyll protoplasts was not induced. Six embryoids were obtained and three lines regenerated to complete plants through embryogenesis. Two of the regenerated lines exhibited intermediate morphological characteristics of the parents in the leaf shape. Chromosome counts showed that these regenerated plants had expected 36 chromosomes (2n=2x=18 for each parent). The rDNA analysis using biotin-labeled rRNA probes confirmed the presence of genomes from both parents in these plants. This somatic hybridization system would be useful for the practical Citrus breeding.     

Cloning and expression of a putative cyclodextrin glucosyltransferase from the symbiotically competent cyanobacterium Nostoc sp. PCC 9229

A polymerase chain reaction-based method was used to isolate a Nostoc sp. PCC 9229 cDNA from infected glands of Gunnera chilensis. The complete gene sequence was isolated from a genomic Nostoc sp. PCC 9229 library. Sequence analysis showed 84% amino acid similarity to a putative cyclodextrin glycosyltransferase from Nostoc sp. PCC 7120 and the gene was therefore termed cgt. Southern blot revealed that the cgt gene was present in symbiotically competent cyanobacteria. The cgt gene was expressed in free-living nitrogen-fixing cultures in light or in darkness when supplemented with fructose. This is the first expression analysis of a cgt gene from a cyanobacterium.     

Nonhost status of Citrus sinensis cultivar valencia and C. paradisi cultivar ruby red to Mexican Anastrepha fraterculus (Diptera: Tephritidae)

Anastrepha fraterculus (Wiedemann) is recognized as a pest of citrus, apples, and blackberries in South America. In Mexico, it is mainly found in fruit of the family Myrtaceae and has never been reported infesting citrus. Here, we sought to determine whether females stemming from Mexican A. fraterculus populations (collected in the state of Veracruz) would lay eggs in 'Valencia' oranges and 'Ruby Red' grapefruit and, if so, whether larvae would hatch and develop. We worked under laboratory and seminatural conditions (i.e., gravid females released in fruit-bearing, bagged branches in a commercial citrus grove) and used Anastrepha ludens (Loew), a notorious pest of citrus, as a control species. Under laboratory conditions, A. ludens readily accepted both oranges and grapefruit as oviposition substrates, but A. fraterculus rarely oviposited in these fruit (but did so in guavas, a preferred host) and no larvae ever developed. Eggs were deposited in the toxic flavedo (A. fraterculus) and nontoxic albedo (A. ludens) regions. Field studies revealed that, as was the case in the laboratory, A. fraterculus rarely oviposited into oranges or grapefruit and that, when such was the case, either no larvae developed (oranges) or of the few (13) that developed and pupated (grapefruit), only two adults emerged that survived 1 and 3 d, respectively (5-17% of the time necessary to reach sexual maturity). In sharp contrast, grapefruit exposed to A. ludens yielded up to 937 pupae and adults survived for >6 mo. Therefore, the inability of Mexican A. fraterculus to successfully develop in citrus renders the status of Mexican A. fraterculus as a pest of citrus in Mexico as unsubstantiated.     

Hepatocyte growth factor (HGF) receptor expression and role of HGF during embryonic mouse testis development

The hepatocyte growth factor (HGF) receptor, c-met, transduces the HGF multiple biological activities. During embryonic development the system HGF/c-met regulates the morphogenesis of different organs and tissues. In this study we examined c-met gene expression during mouse testis development and, by means of Northern blot and in situ hybridization, we report the receptor expression pattern. C-met expression is not detectable in male genital ridges isolated from embryos at 11.5 days postcoitum (dpc). In testes isolated from 12.5 and 13.5 dpc, c-met expression is detectable and essentially localized in the developing cords. Male genital ducts do not express c-met at the reported ages, whereas female ducts appear c-met positive. Moreover, we report that HGF is able to induce testicular morphogenesis in vitro. Male genital ridges isolated from embryos at 11.5 dpc are morphologically nonorganized. Culturing 11.5 dpc urogenital ridges in the presence of HGF we obtained testis organization and testicular cord formation. Our data demonstrate that c-met is expressed during the beginning period of testis differentiation and that HGF is able to support testicular differentiation in vitro. All these data indicate that this growth factor, besides its role as mitogenic factor, plays a fundamental role during testicular cord formation probably inducing cell migration and/or cell differentiation.     

Localization of putative biochemical messengers during Eisenia foetida (Annelida, Oligochaeta) development

This histochemical-immunohistochemical study was performed on the earthworm Eisenia foetida at different developmental stages, to investigate the presence and distribution of cholinergic molecules (AChE, BuChE, alpha-bungarotoxin-binding sites), several biogenic amines (5HT and catecholamines), and some immunologically-related peptides (somatostatin. FMRF-amide, VIP, substance-P, bombesin). The results showed that the pattern of labelling for the markers is different at different stages. In summary, cholinesterases appeared widely distributed in the early embryonic stages. They then were localized in particular areas of the developing nerve and muscle tissues, whereas in newborn and adult earthworms they were restricted to ventral muscular fibers and to some CNS cells. Biogenic amines were constantly present in the embryonic and adult nervous tissues. Immunologically-related peptides were detectable after organogenesis. Our results provide indirect evidence for a role of cholinesterases in regulating early intercellular communications, neurogenesis and myogenesis, and support the hypothesis that some conservative sequences of messenger peptides arose very early in evolution.     

Hordein-gene expression during development of the barley (Hordeum vulgare) endosperm.

A previous study [Rahman, Shewry & Miflin (1982) J. Exp. Bot. 33, 717-728] showed differential accumulation of the major storage proteins (called B and C hordeins) in developing endosperms of barley (Hordeum vulgare). To determine how this accumulation is regulated, we have studied mRNA fractions prepared from similar endosperms. Hordein-related mRNA species were detected some days before the deposition of hordeins in vivo. The translation products in vivo directed by polyribosomes, polysomal RNA and total cellular RNA showed similar changes in the proportions of the hordein products to those observed in the accumulations of the proteins in vivo. There was a relative increase in one of the subfamilies of B hordeins (called B1 hordein) and a decrease in the second subfamily of B hordeins (B3 hordein) and in C hordeins. The populations of RNA species related to these three groups of hordeins were studied by 'dot hybridization', with specific complementary-DNA probes for B1-, B3- and C-hordein-related sequences. This showed a 10-15-fold increase in sequences related to the B1 hordein during endosperm development, but only a 4-fold increase in sequences related to B3 and C hordeins. These results indicate that the rates of synthesis of hordeins are related to the abundance of their respective mRNA species. The different results observed for the two subfamilies of B hordeins are of interest, since they indicate differential expression of two subfamilies of genes present at a single multigenic locus.     

Characterization of mouse thrombospondin 2 sequence and expression during cell growth and development.

Thrombospondin (TSP) is an extracellular matrix glycoprotein whose expression has been associated with a variety of cellular processes including growth and embryogenesis. The recent discovery of the existence of a second mouse TSP gene necessitates careful examination of the discrete biochemical and functional properties associated with each molecule. In this report, the primary structures of human TSP, mouse TSP1 (mTSP1), mouse TSP2 (mTSP2), and chicken TSP are compared; and the expression of mTSP1 and mTSP2 during embryogenesis and growth factor-mediated cell proliferation is examined. The cloning and sequencing of the entire coding regions of mTSP1 and mTSP2 revealed considerable conservation of residues critical for TSP structure and function; these data suggest that TSP2 is capable of trimer formation and many of the same cell-surface and ligand interactions that mediate TSP function. Comparison of the various TSP sequences also allowed the assignment based on sequence homology of previously reported human TSP as TSP1 and chicken TSP as TSP2. mTSP2, like mTSP1, was shown to be a primary response gene when quiescent Swiss 3T3 cells were stimulated with serum, platelet-derived growth factor BB, basic fibroblast growth factor, or interleukin-1 beta. Interestingly, TSP1 and TSP2 exhibited markedly different tissue- and stage-specific patterns of mRNA expression during mouse embryogenesis, implying that the two TSP molecules possess discrete functional properties important for development. Additionally, the TSP genes (Thbs1 and Thbs2) were mapped to single loci on mouse chromosomes 2 and 17, respectively.     

Refinement of the Citrus Tristeza Virus Resistance Gene (Ctv) Positional Map in Poncirus trifoliata and Generation of Transgenic Grapefruit (Citrus paradisi) Plant Lines with Candidate Resistance Genes in this Region

Citrus tristeza virus (CTV) is a major pathogen of Citrus. A single dominant gene Ctv present in the trifoliate relative of Citrus, Poncirus trifoliata confers broad spectrum resistance against CTV. Refinement of genetic maps has delimited this gene to a 121 kb region, comprising of ten candidate Ctv resistance genes. The ten candidate genes were individually cloned in Agrobacterium based binary vector and transformed into three CTV susceptible grapefruit varieties. Two of the candidate R-genes, R-2 and R-3 are exclusively expressed in transgenic plants and in Poncirus trifoliata, while five other genes are also expressed in non-transformed Citrus controls. Northern blotting with a CTV derived probe for assessment of infection in virus inoculated plants over a span of three growth periods, each comprising of six to eight weeks, indicates either an absence of initiation of infection or it’s slow spread in R-2 plant lines or an initial appearance of infection and it’s subsequent obliteration in some R-1 and R-4 plant lines. Limited genome walk up- and downstream form R-1 gene, based on it’s 100% sequence identity between Poncirus and Citrus, indicates promoter identity of 92% between the two varieties. Further upstream and downstream sequencing indicates the presence of an O-methyl transferase and a Copia like gene respectively in Citrus instead of the amino acid transporter like gene upstream and a sugar transporter like gene downstream in Poncirus. The possibility of recombinations in the resistance locus of Citrus and the need for consistent monitoring for virus infection and gene expression in the transgenic Citrus trees is discussed. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Ovine osteopontin: II. Osteopontin and alpha(v)beta(3) integrin expression in the uterus and conceptus during the periimplantation period.

Osteopontin (OPN) is an acidic 70-kDa glycoprotein that is cleaved by proteases to yield 45-kDa and 24-kDa fragments. The 70-kDa and 45-kDa proteins contain a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence that binds to cell surface integrins (primarily alpha(v)beta(3) heterodimer) to promote cell-cell attachment and cell spreading. A 70-kDa acidic protein was previously detected by two-dimensional (2D) PAGE in Day 17 pregnant endometrial cytosolic extracts using Stainsall and identified as immunoreactive OPN using Western blotting. Three forms of immunoreactive OPN proteins (70, 45, and 24 kDa) were detected by 1D PAGE and Western blot analysis of endometrial extracts. OPN protein in endometrial extracts did not differ between cyclic and pregnant ewes. However, the amount of 45-kDa OPN increased in uterine flushings from pregnant ewes between Days 11 and 17. Immunoreactive OPN was localized to luminal and glandular epithelia of both cyclic and pregnant ewes, and to trophectoderm of Day 19 conceptuses. The alpha(v) and beta(3) integrins were detected on Day 19 endometrium and conceptuses by immunofluorescence. It was reported that OPN mRNA increases in the uterine glands of pregnant ewes and secretion of OPN protein into the uterine lumen increases during early pregnancy. The present results demonstrate accumulation of OPN protein on endometrial LE and conceptus trophectoderm. Therefore, it is hypothesized that progesterone and/or interferon-tau induce expression, secretion and/or proteolytic cleavage of OPN by uterine epithelium. Secreted OPN is then available as ligand for alpha(v)beta(3) integrin heterodimer on trophectoderm and uterus to 1) stimulate changes in morphology of conceptus trophectoderm and 2) induce adhesion between luminal epithelium and trophectoderm essential for implantation and placentation.     

Fibroblastic growth factor receptor (FGF-R) expression during uterine involution in goat.

To investigate the possible participation of fibroblastic growth factors (FGFs) in endometrial involution, 20 multiparous goats, slaughtered on days 0, 1, 4, 10, 16 and 22 postpartum (pp), were used. Samples of different parts of the previous pregnant horns were taken and processed using streptoavidin-biotin-peroxidase complex method to analyse FGF receptor (FGF-R) expression. The percentage of positive cells in luminal epithelium, superficial and deep glands and stroma was evaluated. Epithelial, glandular and stromal cells exhibited FGF-R immunoreactivity. No differences between caruncular and inter-caruncular epithelium were observed and staining was most evident in the superficial glands. The greatest degree of FGF-R expression was seen on days 10 and 16 pp, coinciding with epithelial and stromal cellular regeneration. These results suggest that caprine uterine involution is associated with variations in the expression of FGF-R.