Nodulation of specific legumes is controlled by several distinct loci in Rhizobium trifolii

Summary Three distinct loci (designated regions III, IV and V) were identified in the 14 kb Nod region of Rhizobium trifolii strain ANU843 and were found to determine the host range characteristics of this strain. Deletion of region III or region V only from the 14 kb Nod region affected clover nodulation capacity. The introduction to R. Leguminosarum of DNA fragments on multicopy vectors carrying regions III, IV and V (but not smaller fragments) extended the host range of R. leguminosarum so that infection threads and nodules occurred on white clover plants. The same DNA fragments were introduced to the Sym plasmid-cured strain (ANU845) carrying the R. meliloti recombinant nodulation plasmid pRmSL26. Plasmid pRmSL26 alone does not confer root hair curling or nodulation on clover plants. However, the introduction to ANU845 (pRmSL26) of a 1.4 kb fragment carrying R. trifolii region IV only, resulted in the phenotypic activation of marked root hair curling ability to this strain on clovers but no infection events or nodules resulted. Only the transfer of regions III, IV and V to strain ANU845 (pRmSL26) conferred normal nodulation and host range ability of the original wild type R. trifolii strain. These results indicate that the host range genes determine the outcome of early plant-bacterial interactions primarily at the stage of root hair curling and infection.     

Determination of Transgene Copy Number in Stably Transfected Mammalian Cells by PCR-Capillary Electrophoresis Assay

The quantitative determination of transgene copy number in stably transfected mammalian cells has been traditionally estimated by Southern blot analysis. Recently, other methods have become available for appraisal of gene copy number, such as real-time PCR. Herein we describe a new method based on a fluorescently labeled PCR, followed by capillary electrophoresis. We amplified our target gene (prothrombin) and the internal control originating from genomic DNA (18S rRNA) in the same PCR tube and calculated the mean peak height ratio of the target:control gene for every cell clone sample. With this approach we identified stably transfected cell clones bearing the same transgene copy number. The results of our assay were confirmed by real-time PCR. Our method proves to be fast, low-cost, and reproducible compared with traditionally used methods. This assay can be used as a rapid screening tool for the determination of gene copy number in gene expression experiments.     

Molecular characterization of a 21.4 kilobase antibiotic resistance plasmid from an α-hemolytic Escherichia coli O108:H- human clinical isolate

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes.     

Screening of alpha-1-antitrypsin gene by denaturing gradient gel electrophoresis (DGGE)

Alpha-1-antitrypsin (AAT) is a serine protease inhibitor whose deficiency could cause emphysema and liver disease and, as recently described, could be a risk factor for lung cancer development. Alpha-1-antitrypsin inhibits a variety of proteases but its primary target is neutrophil elastase, an extracellular endopeptidase capable of degrading most protein components of the extracellular matrix. Inhibition of neutrophil elastase by AAT has an important role in maintaining the integrity of connective tissue. The gene encoding for AAT spans over 12.2 kb, consists of seven exons and is highly polymorphic. Therefore several methods for mutation screening of alpha-1-antitrypsin gene have been developed. Method described here is based on denaturing gradient gel electrophoresis (DGGE). This method is highly efficient and reliable and allows rapid analysis of entire coding region of alpha-1-antitrypsin gene, including splice junction sites. Previously described DGGE based analysis of AAT gene included overnight electrophoresis of individually amplified fragments. The optimization of the method described in this paper is directed towards the shortening of the duration of electrophoresis and amplification of fragments in multiplex reaction in order to make the analysis less time-consuming and therefore more efficient.     

Ghrelin immunohistochemistry of gastric adenocarcinoma and mucoepidermoid carcinoma of salivary gland

Ghrelin (G-HH) synthesized in several tissues including salivary and stomach glands stimulates appetite in humans by modulating neuropeptide Y neurons in the hypothalamic arcuate nucleus. Loss of appetite is one of the most important symptoms of stomach cancer. We conducted a study using immunohistochemistry to determine whether salivary glands and stomach cancer tissues produce ghrelin. We determined that negative ghrelin immunohistochemistry discriminates tumors from normal tissues and may therefore further our understanding of the clinically important problem of reduced food intake and anorexia in cancer patients. Radioimmunoassay analyses confirmed that cancer cells do not produce a G-HH peptide, whereas normal cells yield this peptide.     

Implications of long-distance flavonoid movement in Arabidopsis thaliana

Flavonoid synthesis is modulated by developmental and environmental signals that control the amounts and localization of the diverse flavonoids found in plants. Flavonoids are implicated in regulating a number of physiological processes including UV protection, fertilization, auxin transport, plant architecture, gravitropism and pathogenic and symbiotic interactions with other organisms. Recently we showed that flavonoids can move long distances in plants, which may facilitate these molecules reaching positions in the plant where these processes are regulated. The localised application of selective flavonoids to tt4 mutants such as naringenin, dihydrokaempferol and dihydroquercetin showed that they were taken up at the root tip, mid-root or cotyledons and travelled long distances via cell-to-cell movement to distal tissues and converted to quercetin and kaempferol. In contrast, kaempferol and quercetin do not move long distances. They were taken up only at the root tip and did not move from this position. Here we show the movement of endogenous flavonoids by using reciprocal grafting experiments between tt4 and wild-type seedlings. These results demonstrated that to understand the distribution of flavonoids in Arabidopsis, it is necessary to know where the flavonoid biosynthetic enzymes are made and to understand the mechanisms by which certain flavonoids move from their site of synthesis.Key words: flavonoid movement, reciprocal graft, quercetin, kaempferol, Arabidopsis thaliana, fluorescence, aglyconeFlavonoids are plant secondary metabolites made by the phenylpropanoid pathway. The central biosynthetic pathway is known and in Arabidopsis most of the enzymes in flavonoid synthesis are encoded by single copy genes.¹ The isolation of mutants with defects in the genes encoding these flavonoid biosynthetic enzymes has allowed researchers to understand the biochemical complexity of flavonoid synthesis and their biological roles. Flavonoid synthesis is more complex in other species, such as legumes, which produce a greater diversity of flavonoid molecules, and in which gene families encode the key enzymatic branch points of the pathway.^2,3The functions of flavonoids were demonstrated using genetic approaches that blocked flavonoid synthesis in Arabidopsis and other species. In Arabidopsis, flavonoids play important roles in UV protection⁴ and regulate auxin transport and dependent physiological processes, such as gravity responses,^5–7 and lateral root formation.⁸ In petunia, maize and tomato, pollen without flavonoids is infertile and this phenotype is reversed by flavonoid addition.^9–11 However, the enigma of why flavonoid-deficient Arabidopsis seedlings are fertile has not been resolved.¹² Flavonoids appear to interact with Multidrug resistance (MDR)/P-glycoproteins (PGP)/ABC-Type B proteins⁷ that transport auxin, regulate phosphatases and kinases, and may have regulatory roles as scavengers of reactive oxygen species (reviewed in ref. ¹³). These results are consistent with a diversity of important functions for flavonoids in plants that require careful control of flavonoid synthesis and localization.We have explored the possibility that flavonoid accumulation in specific locations is also modulated by movement of early intermediates of the flavonoid pathway. Long-distance movement of secondary metabolites is largely unexplored but potentially has profound developmental effects. Grafting experiments conducted in the early 1900s suggested that alkaloids move from the site of manufacture (the root) to the aerial tissue.¹⁴ More recent grafting experiments showed that root synthesised metabolites, perhaps carotenoids, regulate shoot development,^15,16 flowering inducers travel long distances,¹⁷ and phytohormones are translocated (reviewed in ref. ¹⁸).We recently showed that flavonoids moved long distances in Arabidopsis using several approaches.¹⁹ The roots of Arabidopsis seedlings grown in complete darkness do not accumulate flavonoids⁵ since expression of early genes encoding enzymes of flavonoid biosynthesis are light dependent.²⁰ Yet, flavonoids accumulate in root tips of seedlings with light-grown shoots and light-shielded roots, consistent with shoot-to-root flavonoid movement. Using fluorescence microscopy, a selective flavonoid stain (diphenyl boric acid 2-amino ethyl ester [DPBA]), and localised aglycone application to transparent testa mutants, we showed that flavonoids accumulated in tissues distal to the application site, indicating that early intermediates in the flavonoid pathway can move long distances. This was confirmed by time-course fluorescence experiments and HPLC. Flavonoid applications to root tips resulted in basipetal movement in epidermal layers, with subsequent fluorescence detected 1 cm from application sites after 1 h. Flavonoid application mid-root or to cotyledons showed movement of flavonoids toward the root tip mainly in vascular tissue. Naringenin, dihydrokaempferol and dihydroquercetin were taken up at the root tip, mid-root or through cotyledons and travelled long distances via cell-to-cell movement to distal tissues followed by conversion to quercetin and kaempferol. In contrast, kaempferol and quercetin were only taken up at the root tip. Uptake of flavonoids at the root tip was inhibited by glybenclamide, a specific inhibitor of ABC type transporters²¹ suggesting a possible role for transporters of this class in the movement of flavonoids.To show that endogenous flavonoids are capable of long distance movement, we performed reciprocal butt grafting between tt4 and wild-type seedlings.²² In these experiments we asked whether flavonoids moved from wild-type tissues to flavonoid-deficient tissues of tt4. DPBA fluorescence detection was used to detect flavonoid movement into tt4 tissues.¹⁹ Seedlings were grafted and grown on filter paper in Petri dishes for 8 d. The seedlings were then transferred to equal parts sand, perlite and vermiculite to avoid the possibility of uptake of pre-existing flavonoids that may be natural soil components. After 14 d, the seedlings were stained with DPBA. When tt4 roots were grafted to tt4 shoots, the samples showed dim greenish autofluorescence in roots and only red chlorophyll fluorescence in the shoot. When either tt4 roots or shoots were grafted onto wild-type shoots or roots, respectively, the tt4 tissues showed bright yellow DPBA fluorescence (Fig. 1). The results of these experiments clearly showed that endogenous flavonoids moved across the graft to the reciprocal tissue. The flavonoid movement is specific to certain tissues, as flavonoids are not transported into the seeds developing on tt4 shoots grafted on wild-type roots, which retain the transparent testa phenotype. Although flavonoids clearly travelled from wild-type root tissue to mutant shoots, they were not capable of complementing the seed colour defect of tt4. In addition, adding naringenin to tt4 plants either to the media, or to the soil, also did not complement the seed colour phenotype in tt4 (data not shown). Recent research by Hsieh and Huang²³ may account for this inability to complement seed colouration, as flavonoids in the Brassicaceae end up in the pollen coat rather than the testa. The testa tissue derives from ovular tissue,²⁴ and thus is maternal in origin.Open in a separate windowFigure 1Grafting shows flavonoid movement occurs across grafts. Reciprocal grafting between wild type and tt4 indicated flavonoid movement from the flavonoid producing tissue to the chalcone synthase-deficient tissue. The order of the graft is indicated by the left legend as aerial tissue over root tissue in the graft. Micrographs are DPBA stained tissue excited with 488 nm wavelength. The tt4/tt4 control graft shows no flavonoids are present, even though a wound has occurred on the leaf which generally exacerbates flavonoid fluorescence. Scale bar = 100 µm. Green fluorescence is from kaempferol and gold from quercetin. The red fluorescence is from chlorophyll.The complexity of the flavonoid biosynthetic pathway and the large number of modified flavonoids that can be made through the complex series of glycosylation reactions suggests that distinct flavonoid molecules may have unique function. To fully understand these molecules, it is necessary to dissect the synthesis pathways for these glycosylated flavonoids. Two unnamed flavonoid glycoside mutants isolated in 1998,²⁵ have profound developmental phenotypes, supporting this hypothesis. These mutations resulted in whorled cauline leaves on inflorescences and double the number of rosette leaves. Our lab is in the process of determining if other phenotypes exist in flavonoid mutants.A critical feature of the observations of flavonoid movement is understanding the biological context of this movement. First, a number of recent studies reported physiological functions of flavonoids in roots, ranging from modulation of auxin transport and root gravitropism,^5–8 to nodulation³ and root branching,⁸ while it is clear that flavonoid synthesis is absent in dark-grown seedlings.⁵ Yet, for flavonoids to function in roots of plants grown in soil, the light signal and/or flavonoid precursors must travel to the roots. Additionally, transient flavonoid accumulation has been reported in roots reoriented relative to the gravity vector.⁶ For flavonoids to transiently accumulate at the root tip (at 2 hours after reorientation) and to return to lower levels (within 2 additional hours), suggests that more than flavonoid synthesis is regulated. Perhaps this transient flavonoid accumulation requires localized enzyme activation and transport mechanisms. As flavonoid transport is inhibited by a compound that blocks ABC transporters, which include the newly identified auxin transporters of the MDR/PGP class, perhaps there are connections between flavonoid and auxin transport that allow this transient accumulation. A more detailed understanding of this role of flavonoid movement in controlling plant development awaits additional experimentation.     

Flavonoids:New Roles for Old Molecules

Flavonoids are ubiquitous in the plant kingdom and have many diverse functions including defense, UV protection, auxin transport inhibition, allelopathy, and flower coloring. Interestingly, these compounds also have considerable biological activity in plant, animal and bacterial systems – such broad activity is accomplished by few compounds. Yet, for all the research over the last three decades, many of the cellular targets of these secondary metabolites are unknown. The many mutants available in model plant species such as Arabidopsis thaliana and Medicago truncatula are enabling the intricacies of the physiology of these compounds to be deduced. In the present review, we cover recent advances in flavonoid research, discuss deficiencies in our understanding of the physiological processes, and suggest approaches to identify the cellular targets of flavonoids.