Altered profile of secondary metabolites in the root exudates of Arabidopsis ATP-binding cassette transporter mutants

Following recent indirect evidence suggesting a role for ATP-binding cassette (ABC) transporters in root exudation of phytochemicals, we identified 25 ABC transporter genes highly expressed in the root cells most likely to be involved in secretion processes. Of these 25 genes, we also selected six full-length ABC transporters and a half-size transporter for in-depth molecular and biochemical analyses. We compared the exuded root phytochemical profiles of these seven ABC transporter mutants to those of the wild type. There were three nonpolar phytochemicals missing in various ABC transporter mutants compared to the wild type when the samples were analyzed by high-performance liquid chromatography-mass spectrometry. These data suggest that more than one ABC transporter can be involved in the secretion of a given phytochemical and that a transporter can be involved in the secretion of more than one secondary metabolite. The primary and secondary metabolites present in the root exudates of the mutants were also analyzed by gas chromatography-mass spectrometry, which allowed for the identification of groups of compounds differentially found in some of the mutants compared to the wild type. For instance, the mutant Atpdr6 secreted a lower level of organic acids and Atmrp2 secreted a higher level of amino acids as compared to the wild type. We conclude that the release of phytochemicals by roots is partially controlled by ABC transporters.     

The molecular basis of shoot responses of maize seedlings to Trichoderma harzianum T22 inoculation of the root: a proteomic approach

Trichoderma spp. are effective biocontrol agents for several soil-borne plant pathogens, and some are also known for their abilities to enhance systemic resistance to plant diseases and overall plant growth. Root colonization with Trichoderma harzianum Rifai strain 22 (T22) induces large changes in the proteome of shoots of maize (Zea mays) seedlings, even though T22 is present only on roots. We chose a proteomic approach to analyze those changes and identify pathways and genes that are involved in these processes. We used two-dimensional gel electrophoresis to identify proteins that are differentially expressed in response to colonization of maize plants with T22. Up- or down-regulated spots were subjected to tryptic digestion followed by identification using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry and nanospray ion-trap tandem mass spectrometry. We identified 91 out of 114 up-regulated and 30 out of 50 down-regulated proteins in the shoots. Classification of these revealed that a large portion of the up-regulated proteins are involved in carbohydrate metabolism and some were photosynthesis or stress related. Increased photosynthesis should have resulted in increased starch accumulation in seedlings and did indeed occur. In addition, numerous proteins induced in response to Trichoderma were those involved in stress and defense responses. Other processes that were up-regulated were amino acid metabolism, cell wall metabolism, and genetic information processing. Conversely, while the proteins involved in the pathways noted above were generally up-regulated, proteins involved in other processes such as secondary metabolism and protein biosynthesis were generally not affected. Up-regulation of carbohydrate metabolism and resistance responses may correspond to the enhanced growth response and induced resistance, respectively, conferred by the Trichoderma inoculation.     

Liver copper content of rats hypo- or hyperresponsive to dietary cholesterol

The question addressed is whether cholesterol intake reduces the hepatic copper content in rats. For this purpose we have compared the hepatic copper content of two selected rat inbred strains after feeding the animals a control or a high fat, high cholesterol diet. One strain was dietary cholesterol resistant (SHR/OlaIpcv), whereas the other strain was susceptible to dietary cholesterol (BN-Lx/Cub). Dietary cholesterol-susceptible rats have a lower baseline hepatic copper content when compared with their resistant counterparts. The consumption of a hypercholesterolemic diet decreased the liver copper concentration (expressed in microg/g dry weight) to about the same extent in both strains. However, dietary cholesterol did not reduce the absolute (expressed as microg/whole liver) and relative (expressed as microg/whole liver/100 g body weight) copper store of rats. The decrease of liver copper concentration after the high fat, high cholesterol diet is probably not caused by a decrease in whole hepatic copper content, but rather due to dietary-induced hepatomegaly.     

Single-stranded conformation polymorphism (SSCP)-driven indirect sequencing in detection of short deletion

To seek for novel rare and/or sporadic mutations within genomic neighborhood of common −344 C>T (rs2427827) FCER1A proximal promoter polymorphism, which by its prevalence could have masked the presence of less frequent genetic variants in our previous single-stranded conformational polymorphism (SSCP) mutational search study, SSCP screening was repeated using primers fixing −344 C>T variant. The genomic region surrounding −344 C>T polymorphism was confirmed to be fairly conservative and only a single sporadic mutation (present in 1 out of 196 chromosomes), a 6-bp deletion −262 to 257 del CTAGAC, was found. From the methodological point of view, we demonstrated a successful detection of a short-length polymorphism using SSCP screening in a population, in which the same genomic region contained frequent single-nucleotide polymorphic variant. In conjunction with subsequent cloning of aberrantly migrating PCR products and SSCP-driven indirect sequencing of the clones, this method offers a superior (to direct sequencing of PCR products) detection of rare mutations. The cost-effective method applied by us enables a reliable characterization of infrequent (thus present only in heterozygotic form) short-length variance of the sequence which are difficult to interpret by direct sequencing.     

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life

N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide.     

Does anthocyanin degradation play a significant role in determining pigment concentration in plants?

In contrast to the detailed knowledge available on anthocyanin synthesis, very little is known about its stability and catabolism in plants. Here we review evidence supporting in planta turnover and degradation of anthocyanins. Transient anthocyanin accumulation and disappearance during plant development or changes in environmental conditions suggest that anthocyanin degradation is controlled and induced when beneficial to the plant. Several enzymes have been isolated that degrade anthocyanins in postharvest fruit that may be candidates for in vivo degradation. Three enzyme groups that control degradation rates of anthocyanins in fruit extracts and juices are polyphenol oxidases, peroxidases and β-glucosidases. Evidence supporting the involvement of peroxidases and β-glucosidases in in vivo anthocyanin degradation in Brunfelsia flowers is presented. Understanding the in vivo anthocyanin degradation process has potential for enabling increased pigmentation and prevention of color degradation in crops.     

The trigger for barn owl (Tyto alba) attack is the onset of stopping or progressing of the prey

Aiming at the question of whether barn owls favor to strike a moving or a stationary prey, we scrutinized the timing of launching 50 attacks of five barn owls (10 attacks/owl) in a captive environment. Attacks on stationary voles outnumbered attacks on moving voles, but this could merely reflect the higher portion of time during which voles were stationary. Notably, the majority of attacks (85-100%) were launched in less than 2 s (measured at accuracy of 0.04 s) after the voles ceased to progress or initiated progression. Therefore, the trigger for launching an attack is the transition of the prey from locomotion to still posture or vice versa.     

Micropropagation of Wild Service Tree (Sorbus torminalis [L.] Crantz): The Regulative Role of Different Aromatic Cytokinins During Organogenesis

The influences of three different aromatic cytokinin derivatives [6-benzylaminopurine, meta-topolin, and 6-(3-methoxybenzylamino)purine-9-ß-D-ribofuranoside (MeOBAPR)] on in vitro multiplication and rhizogenesis of the wild service tree (Sorbus torminalis [L.] Crantz) were compared. The highest micropropagation rate (24 new shoots per explant after 3 months of cultivation) was achieved on media containing BAP. On the other hand, the best rooting microcuttings were those multiplied on a medium containing MeoBAPR. To compare these results with the levels of endogenous cytokinins in multiplied explants, a newly developed UPLC-ESI(+)-MS/MS method was used to determine levels of 50 cytokinin metabolites in explants cultivated 12 weeks on media supplemented by BAP and of the two other aromatic cytokinin analogs used. Several significant differences among the levels of endogenous cytokinins, extracted from the explants, were found. The concentration of BAP9G, an important metabolite suspected to be responsible for inhibition of rooting and acclimatization problems of newly formed plantlets, was found to be the highest in microcuttings grown on media supplemented with BAP. This agrees well with the results of our rooting experiments; the lowest percentages of rooted plantlets 6 weeks after transferring shoots on rooting medium were present on explants multiplied on BAP. In contrast, BAP was still the most effective for the induction of bud formation on primary explants. Levels of the most active endogenous isoprenoid cytokinins, tZ, tZR, and iPR, as well as O-glucosides were also suppressed in explants grown on BAP compared with those of explants treated with other cytokinin derivatives. This may be the result of a very high BAP uptake into the explants grown on this cytokinin. On the other hand, endogenous concentrations of cis-zeatin derivatives as well as dihydrozeatin derivatives were not affected. Differences in the production of another plant hormone, ethylene, that plays an important role in controlling organogenesis in tissue culture, were also observed among S. torminalis plantlets grown in vitro on media containing different cytokinins tested. The highest ethylene levels were detected in the vessels containing media supplemented with mT. They were two to four times higher compared with the production by the S. torminalis explants cultivated on other media used. Finally, the levels of free IAA were also determined in the explants. S. torminalis plantlets grown on media containing BAP contained the lowest level of auxin, which is again in good agreement with their loss of rooting capacity. The results found in this study about optimal plant hormone concentrations may be used to improve in vitro rooting efficiency of the wild service tree and possibly also of other plant species.