Highly efficient <Emphasis Type="Italic">in vitro</Emphasis> adventitious shoot regeneration of peppermint (<Emphasis Type="Italic">Mentha</Emphasis> x <Emphasis Type="Italic">piperita</Emphasis> L.) using internodal explants

An in vitro regeneration system with a 100% efficiency rate was developed in peppermint [Mentha x piperita] using 5- to 7-mm-long second internode stem segments of 3-wk-old stock plants. Shoots developed at sites of excision on stem fragments either directly from the cells or via primary calluses. The optimal medium for maximum shoot initiation and regeneration contained Murashige and Skoog (MS) salts, B5 vitamins, thidiazuron (TDZ, 11.35 μM), ZT (4.54 μM), 10% coconut water (CW), 20 g l⁻¹ sucrose, 0.75% agar, adjusted to pH 5.8. A frequency of 100% shoot initiation was achieved, with an average of 39 shoots per explant. This regeneration system is highly reproducible. The regenerated plants developed normally and were phenotypically similar to Black Mitcham parents.     

Occasional intraguild predation structuring small mammal assemblages: the marsupial Didelphis aurita in the Atlantic Forest of Brazil

The didelphid marsupial, Didelphis aurita, is suggested as an intraguild predator and as key‐species in small mammal assemblages of the Atlantic Forest of Brazil. The field experiments required to test this hypothesis are complex to implement, but the recent revival of regression methods offers a viable alternative. Here we use the dynamic and static regression methods to determine the importance of D. aurita as a competitor and intraguild predator. Capture–recapture data from two localities in the Rio de Janeiro State were used, Garrafão (municipality of Guapimirim), a coastal forest of the Serra do Mar, and Barra de Maricá, a costal sand dune vegetation. Population and microhabitat variables were monitored from April 1997 to April 2003 in Garrafão, and from January 1986 to July 1990 in Barra de Maricá. Microhabitat variables were related to Canopy, Plant, Litter and Rock covers, Obstruction from 0 to 1.5 m, and Number of logs. Exploitation competition was tested by the dynamic method, which models the effects of D. aurita on the per capita growth rate of a species. Interference by predation or competition was tested by the static method, where the abundance of D. aurita at trap stations was regressed against the abundance of other small mammals, after removal of any variation associated with microhabitat factors. Exploitation competition was not detected, but the interference of D. aurita was pervasive, affecting all small mammals studied in the two localities. The clear avoidance of D. aurita by all small mammals tested in two localities of different physiognomies indicates that it functions as an intraguild predator, even if actual predation by D. aurita is an occasional event.     

Histone hyperacetylation affects meiotic recombination and chromosome segregation in Arabidopsis

In this study, the meiotic role of MEIOTIC CONTROL OF CROSSOVERS1 (MCC1), a GCN5‐related histone N‐acetyltransferase, is described in Arabidopsis. Analysis of the over‐expression mutant obtained by enhancer activation tagging revealed that acetylation of histone H3 increased in male prophase I. MCC1 appeared to be required in meiosis for normal chiasma number and distribution and for chromosome segregation. Overall, elevated MCC1 did not affect crossover number per cell, but has a differential effect on individual chromosomes elevating COs for chromosome 4, in which there is also a shift in chiasma distribution, and reducing COs for chromosome 1 and 2. For the latter there is a loss of the obligate CO/chiasma in 8% of the male meiocytes. The meiotic defects led to abortion in about half of the male and female gametes in the mutant. In wild type, the treatment with trichostatin A, an inhibitor of histone deacetylases, phenocopies MCC1 over‐expression in meiosis. Our results provide evidence that histone hyperacetylation has a significant impact on the plant meiosis.     

Long-term trends of heron and egret populations in Italy, and the effects of climate, human-induced mortality, and habitat on population dynamics

Factors affecting bird population dynamics include climate, harvesting by humans, and habitat changes. Here, we describe the long-term (1972–2006) population trends of seven heron species in NW Italy, an area holding important European breeding populations of these species. Grey (Ardea cinerea), purple (Ardea purpurea), and squacco (Ardeola ralloides) herons, and little egrets (Egretta garzetta) exhibited a strong logistic increase, leveling off around year 2000 at 3–23 times their initial level. Black-crowned night herons (Nycticorax nycticorax) began by increasing like the former species but then dropped to initial levels. Such trends were found to be influenced by several candidate ecological factors, as assessed by ARIMA models. Specifically, grey herons increased following a decrease in human-induced mortality, as quantified by an index of hunting pressure, and an increase in winter temperatures. Little egrets increased mainly with the increase of the extent of ricefields, whereas squacco herons increased with increasing rainfall in the African wintering range. Black-crowned night herons were also positively affected by increasing African rainfall, but only during 1972–1988, whereas in later years competition with other herons could have affected the species’ decline. The improved protection of colony sites by special reserves was unlikely to be the primary trigger of the observed increase, although obviously important for the long-term population persistence. In conclusion, our study shows that heron populations of southern Europe are sensitive to environmental and climatic changes, as well as to temporal variation in human disturbance and changes in foraging habitats, though the importance of the different factors differs among species.     

Solution structure of the human signaling protein RACK1

Background  

The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development.     

Use of the Plant Defense Protein Osmotin To Identify Fusarium oxysporum Genes That Control Cell Wall Properties

Fusarium oxysporum is the causative agent of fungal wilt disease in a variety of crops. The capacity of a fungal pathogen such as F. oxysporum f. sp. nicotianae to establish infection on its tobacco (Nicotiana tabacum) host depends in part on its capacity to evade the toxicity of tobacco defense proteins, such as osmotin. Fusarium genes that control resistance to osmotin would therefore reflect coevolutionary pressures and include genes that control mutual recognition, avoidance, and detoxification. We identified FOR (Fusarium Osmotin Resistance) genes on the basis of their ability to confer osmotin resistance to an osmotin-sensitive strain of Saccharomyces cerevisiae. FOR1 encodes a putative cell wall glycoprotein. FOR2 encodes the structural gene for glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting step in the biosynthesis of hexosamine and cell wall chitin. FOR3 encodes a homolog of SSD1, which controls cell wall composition, longevity, and virulence in S. cerevisiae. A for3 null mutation increased osmotin sensitivity of conidia and hyphae of F. oxysporum f. sp. nicotianae and also reduced cell wall β-1,3-glucan content. Together our findings show that conserved fungal genes that determine cell wall properties play a crucial role in regulating fungal susceptibility to the plant defense protein osmotin.Studies of plant-pathogen interactions strongly suggest that under the pressure to survive, plants and pathogens continuously react to one another''s defense arsenal and evolve to overcome these defenses (13). Plants recognize pathogen-associated molecular patterns, such as fungal cell wall fragments composed of chitin, glucans, oligosaccharides, or glycoprotein peptides (32). It has been established that pathogens evolved effector proteins to avoid plant surveillance mechanisms that recognize pathogen-associated molecular patterns and this in turn led to the evolution of plant surveillance mechanisms that recognize pathogen-specific effector proteins. All pathogen recognition mechanisms induce intracellular signaling that culminates in the synthesis of factors, such as antimicrobial plant proteins, that help in limiting the severity of infection (74). The antimicrobial proteins are therefore among the ultimate effectors of plant defense. There is evidence of recognition between plant antimicrobial proteins and pathogen-specific molecules (74). Therefore, pathogen mechanisms of resistance to the antimicrobial proteins and the antimicrobial proteins themselves must have coevolved. Consequently, we postulated that a screen for fungal genes that alter the sensitivity of a phytopathogen to an antifungal protein of the host plant (that is, a cognate plant defense effector) would lead to identification of genes involved in controlling pathogenicity, in controlling access of the antifungal protein to its target fungal molecules (such as genes controlling cell surface composition), and in controlling detoxification mechanisms.The plant antifungal protein selected to test this hypothesis was osmotin. Osmotin is an antifungal protein that is overexpressed in and secreted by salt-adapted cultured tobacco (Nicotiana tabacum) cells (63). It is a member of a family of ubiquitous plant proteins, referred to as plant pathogenesis-related proteins of family 5 (PR-5), that are implicated in defense against fungi (74). Osmotin gene and protein expression is induced by biotic stresses, and overexpression of osmotin delays development of disease symptoms in transgenic plants (41, 42, 43, 84). The genetic bases of the susceptibility and resistance of Saccharomyces cerevisiae to osmotin have been explored in our laboratory (49, 50). The results show that specific interactions of osmotin with the plasma membrane are responsible for cell death signaling. However, because the cell wall governs access of osmotin to the plasma membrane, differences in cell wall composition largely account for the differential osmotin sensitivity of various S. cerevisiae strains, and specific cell wall components play a significant role in modulating osmotin toxicity (30, 31, 49, 50, 81, 82). These studies in the model nonpathogenic fungus, S. cerevisiae, support our hypothesis that a screen for genes that alter the sensitivity of a phytopathogenic fungus to an antifungal defense effector protein of the host plant will uncover genes involved in controlling access of the antifungal protein to its target fungal molecules.Osmotin, like other plant defense antifungal proteins, has specific but broad-spectrum antifungal activity (74). One of the most osmotin-sensitive phytopathogenic fungi is Fusarium oxysporum. F. oxysporum is an ascomycete fungus, like S. cerevisiae, and has been touted as an appropriate multihost model for studying fungal virulence (53). It is a soilborne plant pathogen of economic significance, because it causes vascular wilt disease on a large variety of crop plants and produces toxic food contaminants (17, 58). In humans it also causes skin, nail, and eye disease that can become serious or life-threatening illnesses in immunocompromised patients (52, 69). F. oxysporum f. sp. lycopersici, F. oxysporum f. sp. nicotianae, and F. oxysporum f. sp. meloni, like S. cerevisiae, are quite sensitive to osmotin (1, 51; M. L. Narasimhan, unpublished data). Furthermore, it was recently shown that overexpression in F. oxysporum f. sp. nicotianae of an S. cerevisiae cell wall glycoprotein that increases the osmotin resistance of S. cerevisiae also increases the osmotin resistance of the plant pathogen and its virulence on tobacco, the osmotin-producing host plant (51). This suggested that osmotin resistance mechanisms may be conserved between S. cerevisiae and F. oxysporum and that S. cerevisiae could be used as a tool to uncover F. oxysporum genes that control osmotin sensitivity or resistance.In the current study, we expressed an F. oxysporum f. sp. nicotianae cDNA library in the osmotin-sensitive S. cerevisiae strain BWG1-7a and selected genes for their ability to increase osmotin tolerance. We report here the identification and characterization of three FOR (Fusarium Osmotin Resistance) genes that affect the cell wall in S. cerevisiae. The product of FOR1 has homology with a putative cell surface glycoprotein; FOR2 encodes glutamine:fructose-6-phosphate amidotransferase (GFAT), an enzyme that catalyzes the first step in the biosynthetic pathway leading to amino sugar-containing macromolecules, such as glycoproteins and chitin (64); and FOR3 has high homology with S. cerevisiae SSD1, a gene that controls cell wall composition and virulence (31, 78). FOR2 and FOR3 are the functional equivalents of the corresponding S. cerevisiae genes. Our parallel analysis using two model fungi verifies the notion that cell wall proteins play a critical role in determining the sensitivity/resistance of fungi to osmotin. In addition, these results implicate that the tobacco defense protein, osmotin, can serve as an effective/useful tool in identifying genes that control cell wall composition not only in a model fungus, such as S. cerevisiae, but also in phytopathogenic fungi, such as F. oxysporum.     

A spectroscopic analysis of the interaction between the human regulatory proteins RACK1 and Ki-1/57

Ki-1/57 is a 57-kDa cytoplasmic and nuclear protein associated with protein kinase activity and is hyper-phosphorylated on Ser/Thr residues upon cellular activation. In previous studies we identified the receptor of activated kinase-1 (RACK1), a signaling adaptor protein that binds activated PKC, as a protein that interacts with Ki-1/57. Here we demonstrate that the far-UV circular dichroism spectrum of the WD repeat-containing RACK1 protein shows an unusual positive ellipticity at 229 nm, which in other proteins of the WD family has been attributed to surface tryptophans that are quenchable by N-bromosuccinimide (NBS). As well as NBS, in vitro binding of 6xHis-Ki-1/57(122-413) and 6xHis-Ki-1/57(264-413) can also quench the positive ellipticity of the RACK1 spectrum. We generated a model of RACK1 by homology modeling using a G protein beta subunit as template. Our model suggests the family-typical seven-bladed beta-propeller, with an aromatic cluster around the central tunnel that contains four Trp residues (17, 83, 150, 170), which are likely involved in the interaction with Ki-1/57.     

toxin-mediated insect resistance in plants

We are currently in an interesting phase of plant biotechnology releases, both for the scientists responsible for these innovations who are beginning to see their ideas realized, and for the biotechnology companies that are starting to see a return on their investment. One of the most notable examples, is the introduction of transgenic crops that are engineered to express a Bacillus thuringiensis toxin that confers resistance to insect predation. However, the picture is not altogether positive - there is concern that the introduction of this technology was premature or should not have happened at all, and that the valuable insecticidal properties of Bacillus thuringiensis will be lost.     

Identification of regions of the tomato gamma-glutamyl kinase that are involved in allosteric regulation by proline

The first step of proline biosynthesis is catalyzed by gamma-glutamyl kinase (GK). To better understand the feedback inhibition properties of GK, we randomly mutagenized a plasmid carrying tomato tomPRO1 cDNA, which encodes proline-sensitive GK. A pool of mutagenized plasmids was transformed into an Escherichia coli GK mutant, and proline-overproducing derivatives were selected on minimal medium containing the toxic proline analog 3,4-dehydro-dl-proline. Thirty-two mutations that conferred 3,4-dehydro-dl-proline resistance were obtained. Thirteen different single amino acid substitutions were identified at nine different residues. The residues were distributed throughout the N-terminal two-thirds of the polypeptide, but 9 mutations affecting 6 residues were in a cluster of 16 residues. GK assays revealed that these amino acid substitutions caused varying degrees of diminished sensitivity to proline feedback inhibition and also resulted in a range of increased proline accumulation in vivo. GK belongs to a family of amino acid kinases, and a predicted three-dimensional model of this enzyme was constructed on the basis of the crystal structures of three related kinases. In the model, residues that were identified as important for allosteric control were located close to each other, suggesting that they may contribute to the structure of a proline binding site. The putative allosteric binding site partially overlaps the dimerization and substrate binding domains, suggesting that the allosteric regulation of GK may involve a direct structural interaction between the proline binding site and the dimerization and catalytic domains.