Strigolactones: a novel class of phytohormones that inhibit the growth and survival of breast cancer cells and breast cancer stem-like enriched mammosphere cells.

Several naturally occurring phytohormones have shown enormous potential in the prevention and treatment of variety of different type of cancers. Strigolactones (SLs) are a novel class of plant hormones produced in roots and regulate new above ground shoot branching, by inhibiting self-renewal of undifferentiated meristem cells. Here, we study the effects of six synthetic SL analogs on breast cancer cell lines growth and survival. We show that SL analogs are able to inhibit proliferation and induce apoptosis of breast cancer cells but to a much lesser extent "non-cancer" lines. Given the therapeutic problem of cancer recurrence which is hypothesized to be due to drug resistant cancer stem cells, we also tested the ability of SL analogs to inhibit the growth of mammosphere cultures that are typically enriched with cancer stem-like cells. We show that SLs are potent inhibitors of self-renewal and survival of breast cancer cell lines grown as mammospheres and even a short exposure leads to irreversible effects on mammosphere dissociation and cell death. Immunoblot analysis revealed that SLs analogs induce activation of the stress response mediated by both P38 and JNK1/2 MAPK modules and inhibits PI3K/AKT activation. Taken together this study indicates that SLs may be promising anticancer agents whose activities may be achieved through modulation of stress and survival signaling pathways.

Identification of a novel genetically controlled step in mycorrhizal colonization: plant resistance to infection by fungal spores but not extra-radical hyphae.

Vesicular arbuscular mycorrhizal fungi infect plants by means of both spores and vegetative hyphae at early stages of symbiosis. Using 2500 M2 fast-neutron-mutagenized seeds of the miniature tomato (Lycopersicon esculentum) cultivar, Micro-Tom, we isolated a mutant, M161, that is able to resist colonization in the presence of Glomus intraradices spores. The myc(-) phenotype of the mutant was stable for nine generations, and found to segregate as a single Mendelian recessive locus. The mutant exhibited morphological and growth-pattern characteristics similar to those of wild-type plants. Alterations of light intensity and day/night temperatures did not eliminate the myc(-) characteristic. Resistance to mycorrhizal fungal infection and colonization was also evident following inoculation with the fungi Glomus mosseae and Gigaspora margarita. Normal colonization of M161 was evident when mutant plants were grown together with arbuscular mycorrhizal-inoculated wild-type plants in the same growth medium. During evaluation of the pre-infection stages in the mutant rhizosphere, spore germination and appressoria formation of G. intraradices were lower by 45 and 70%, respectively, than the rates obtained with wild-type plants. These results reveal a novel, genetically controlled step in the arbuscular mycorrhizal colonization process, governed by at least one gene, which significantly reduces key steps in pre-mycorrhizal infection stages.

The defense response elicited by the pathogen Rhizoctonia solani is suppressed by colonization of the AM-fungus Glomus intraradices.

Defense responses of alfalfa roots to the pathogenic fungus Rhizoctonia solani were reduced significantly in roots simultaneously infected with the vesicular arbuscular mycorrhizal (AM) fungus Glomus intraradices. R. solani induced five- to tenfold increases in the steady-state levels of chalcone isomerase and isoflavone reductase mRNAs a doubling of root peroxidase activity and a marked autofluorescence in the infected tissue. These changes were inhibited by the presence of G. intraradices. Interestingly, germination of G. intraradices spores and hyphal elongation were sensitive to low concentrations (2 µM) of medicarpin-3-O-glucoside, an isoflavonoid phytoalexin that accumulated both in roots colonized by the pathogenic fungus as well as in AM-treated roots receiving high P, where no colonization by the beneficial fungus occurred. These data support the hypothesis that during early stages of colonization by G. intraradices, suppression of defense-related properties is associated with the successful establishment of AM symbiosis.

Genetic transformation of HeLa cells by Agrobacterium.

Agrobacterium tumefaciens is a soil phytopathogen that elicits neoplastic growths on the host plant species. In nature, however, Agrobacterium also may encounter organisms belonging to other kingdoms such as insects and animals that feed on the infected plants. Can Agrobacterium, then, also infect animal cells? Here, we report that Agrobacterium attaches to and genetically transforms several types of human cells. In stably transformed HeLa cells, the integration event occurred at the right border of the tumor-inducing plasmid's transferred-DNA (T-DNA), suggesting bona fide T-DNA transfer and lending support to the notion that Agrobacterium transforms human cells by a mechanism similar to that which it uses for transformation of plants cells. Collectively, our results suggest that Agrobacterium can transport its T-DNA to human cells and integrate it into their genome.

Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene.

Threonine, lysine, methionine, and tryptophan are essential amino acids for humans and monogastric animals. Many of the commonly used diet formulations, particularly for pigs and poultry, contain limiting amounts of these amino acids. One approach for raising the level of essential amino acids is based on altering the regulation of their biosynthetic pathways in transgenic plants. Here we describe the first production of a transgenic forage plant, alfalfa (Medicago sativa L.) with modified regulation of the aspartate-family amino acid biosynthetic pathway. This was achieved by over-expressing the Escherichia coli feedback-insensitive aspartate kinase (AK) in transgenic plants. These plants showed enhanced levels of both free and protein-bound threonine. In many transgenic plants the rise in free threonine was accompanied by a significant reduction both in aspartate and in glutamate. Our data suggest that in alfalfa, AK might not be the only limiting factor for threonine biosynthesis, and that the free threonine pool in this plant limits its incorporation into plant proteins.

Mycorrhiza-induced changes in disease severity and PR protein expression in tobacco leaves

The development of leaf disease symptoms and the accumulation of pathogenesis-related (PR) proteins were monitored in leaves of tobacco (Nicotiana tabacum cv. Xanthinc) plants colonized by the arbuscular mycorrhizal fungus Glomus intraradices. Leaves of mycorrhizal plants infected with the leaf pathogens Botrytis cinerea or tobacco mosaic virus showed a higher incidence and severity of necrotic lesions than those of nonmycorrhizal controls. Similar plant responses were obtained at both low (0.1 mM) and high (1.0 mM) nutritional P levels and with mutant plants (NahG) that are unable to accumulate salicylic acid. Application of PR-protein activators induced PR-1 and PR-3 expression in leaves of both nonmycorrhizal and mycorrhizal plants; however, accumulation and mRNA steady-site levels of these proteins were lower, and their appearance delayed, in leaves of the mycorrhizal plants. Application of 0.3 mM phosphate to the plants did not mimic the delay in PR expression observed in the mycorrhizal tobacco. Together, these data strongly support the existence of regulatory processes, initiated in the roots of mycorrhizal plants, that modify disease-symptom development and gene expression in their leaves.

Isolation and characterization of two different cDNAs of delta1-pyrroline-5-carboxylate synthase in alfalfa, transcriptionally induced upon salt stress.

Two different cDNA clones, MsP5CS-1 and MsP5CS-2, encoding delta1 -pyrroline-5-carboxylate synthase (P5CS). the first enzyme of the proline biosynthetic pathway, were isolated from a lambdaZap-cDNA library constructed from salt stressed Medicago sativa roots. MsP5CS-1 (2.6 kb) has an open reading frame of 717 amino acids, as well as a non-spliced intron at a position corresponding to the evolutionary fusion point of the bacterial proA and proB genes. MsP5CS-2 (1.25 kb) is a partial clone. The clones share 65% identity in nucleotide sequences, 74% homology in deduced amino acid sequences, and both show a high similarity to Vigna aconitifolia and Arabidopsis thaliana P5CS cDNA clones. Southern blot analysis confirmed the presence of two different P5CS genes. The effect of salinity on the transcription of MsP5CS-1 and MsP5CS-2 in roots was studied, using northern blot analysis and a RT-PCR approach. A rapid increase in the steady-state transcript level of both genes in roots was observed by RT-PCR upon exposure of hydroponically grown 6-day old seedlings to 90 mM NaCl, suggesting that both are salt-inducible genes, yet a higher response was observed for MsP5CS-2.

Signal Transduction Pathways in Mycorrhizal Associations: Comparisons with the Rhizobium-Legume Symbiosis.

A number of genera of soil fungi interact with plant roots to establish symbiotic associations whereby phosphate acquired by the fungus is exchanged for fixed carbon from the plant. Recent progress in investigating these associations, designated as mycorrhizae (sing., mycorrhiza), has led to the identification of specific steps in the establishment of the symbiosis in which the fungus and the plant interact in response to various molecular signals. Some of these signals are conserved with those of the Rhizobium-legume nitrogen-fixing symbiosis, suggesting that the two plant-microbe interactions share a common signal transduction pathway. Nevertheless, only legume hosts nodulate in response to Rhizobium, whereas the vast majority of flowering plants establish mycorrhizal associations. The key questions for the future are: what are the signal molecules produced by mycorrhizal fungi and how are they perceived by the plant? Copyright 1998 Academic Press.

Suppression of tobacco basic chitinase gene expression in response to colonization by the arbuscular mycorrhizal fungus Glomus intraradices.

A differentially displayed cDNA clone (MD17) was isolated from tobacco roots (nicotiana tabacum cv. Xanthi-nc) infected with the arbuscular mycorrhizal (AM) fungus Glomus intraradices. The isolated DNA fragment exhibited a reduced level of expression in response to AM establishment and 90% identity with the 3' noncoding sequence of two basic chitinases (EC 3.2.1.14) from N. tabacum. Northern (RNA) blots and Western blots (immunoblots), probed with tobacco basic chitinase gene-specific probe and polyclonal antibodies raised against the chitinase enzyme, yielded hybridization patterns similar to those of MD17. Moreover, the up-regulation of the 32-kDa basic chitinase gene expression in tobacco roots by (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) was less effective in mycorrhizal roots than in nonmycorrhizal controls. Suppression of endogenous basic chitinase (32-kDa) expression was also observed in transgenic mycorrhizal plants that constitutively express the 34-kDa basic chitinase A isoform. When plants were grown with an increased phosphate supply, no suppression of the 32-kDa basic chitinase was obtained. These findings indicate that during the colonization and establishment of G. intraradices in tobacco roots, expression of the basic chitinase gene is down-regulated at the mRNA level.

The role of EDTA in lead transport and accumulation by indian mustard

Indian mustard (Brassica juncea) plants exposed to Pb and EDTA in hydroponic solution were able to accumulate up to 55 mmol kg-1 Pb in dry shoot tissue (1.1% [w/w]). This represents a 75-fold concentration of Pb in shoot tissue over that in solution. A threshold concentration of EDTA (0.25 mm) was found to be required to stimulate this dramatic accumulation of both Pb and EDTA in shoots. Below this threshold concentration, EDTA also accumulated in shoots but at a reduced rate. Direct measurement of a complex of Pb and EDTA (Pb-EDTA) in xylem exudate of Indian mustard confirmed that the majority of Pb in these plants is transported in coordination with EDTA. The accumulation of EDTA in shoot tissue was also observed to be directly correlated with the accumulation of Pb. Exposure of Indian mustard to high concentrations of Pb and EDTA caused reductions in both the transpiration rate and the shoot water content. The onset of these symptoms was correlated with the presence of free protonated EDTA (H-EDTA) in the hydroponic solution, suggesting that free H-EDTA is more phytotoxic than Pb-EDTA. These studies clearly demonstrate that coordination of Pb transport by EDTA enhances the mobility within the plants of this otherwise insoluble metal ion, allowing plants to accumulate high concentrations of Pb in shoots. The finding that both H-EDTA and Pb-EDTA are mobile within plants also has important implications for the use of metal chelates in plant nutritional research.

Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved.

Transcripts for two genes expressed early in alfalfa nodule development (MsENOD40 and MsENOD2) are found in mycorrhizal roots, but not in noncolonized roots or in roots infected with the fungal pathogen Rhizoctonia solani. These same two early nodulin genes are expressed in uninoculated roots upon application of the cytokinin 6-benzylaminopurine. Correlated with the expression of the two early nodulin genes, we found that mycorrhizal roots contain higher levels of trans-zeatin riboside than nonmycorrhizal roots. These data suggest that there may be conservation of signal transduction pathways between the two symbioses-nitrogen-fixing nodules and phosphate-acquiring mycorrhizae.

Promotion of nod Gene Inducers and Nodulation in Common Bean (Phaseolus vulgaris) Roots Inoculated with Azospirillum brasilense Cd.

Inoculation of Phaseolus vulgaris with Azospirillum brasilense Cd promoted root hair formation in seedling roots and significantly increased total and upper nodule numbers at different concentrations of Rhizobium inoculum. In experiments carried out in a hydroponic system, A. brasilense caused an increase in the secretion of nod gene-inducing flavonoids, as was observed by nod gene induction assays of root exudates fractionated by high-performance liquid chromatography. Possible mechanisms involved in the influence of A. brasilense on this symbiotic system are discussed.

Suppression of an Isoflavonoid Phytoalexin Defense Response in Mycorrhizal Alfalfa Roots.

Isoflavonoids and steady-state mRNA levels of phenylalanine ammonia-lyase, chalcone isomerase, and isoflavone reductase were followed during a rapid, nearly synchronous infection of alfalfa (Medicago sativa L.) roots by the vesicular arbuscular fungus Glomus intraradices (Schenck & Smith) to test whether previously indicated suppression of the host defense response is regulated by changes in the steady-state mRNA level. Relative amounts of steady-state phenylalanine ammonia-lyase mRNA in the mycorrhizal roots doubled between d 14 and 18 and then immediately declined by 75% to reach and maintain a value lower than the control roots through d 21. Relative levels of chalcone isomerase mRNA in the inoculated roots increased 6-fold between d 14 and 17 and then decreased rapidly to the control level. Isoflavone reductase mRNA was not induced by mycorrhizal colonization. High-performance liquid chromatography, proton-nuclear magnetic resonance, and fast atom bombardment-mass spectrometry analyses showed consistent increases in formononetin levels and transient increases in medicarpin-3-O-glycoside and formononetin conjugates in the inoculated roots when colonization began. As colonization increased, levels of formononetin conjugates declined in mycorrhizal roots below those in uncolonized controls. Medicarpin aglycone, an alfalfa phytoalexin normally associated with pathogenic infections, was not detected at any stage. These findings supply detailed evidence that, during early colonization of plant roots by symbiotic Glomus, defense transcripts are induced and then subsequently suppressed.

Plant isoflavonoids, pathogens and symbionts.

It has recently been discovered that when symbiotic Rhizobium and Bradyrhizobium cells are outside the plant they are also exposed to the isoflavonoid phytoalexins that are normally associated with pathogenic infections. How the symbionts elicit and respond to isoflavonoids may help to define the mechanisms that are used by other beneficial soil microorganisms to colonize plant roots.

A Vesicular Arbuscular Mycorrhizal Fungus (Glomus intraradix) Induces a Defense Response in Alfalfa Roots.

Flavonoid accumulation and activities of phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), and chitinase were followed during early colonization of alfalfa roots (Medicago sativa L. cv Gilboa) by vesicular arbuscular (VA) fungi (Glomus intraradix). Formononetin was the only flavonoid detected that showed a consistent increase in the inoculated roots. This increase depended only on the presence of the fungus in the plant rhizosphere; no colonization of the root tissue was required. CHI and chitinase activities increased in inoculated roots prior to colonization, whereas the increase in PAL activity coincided with colonization. After reaching a maximum, activities of all enzymes declined to below those of uninoculated roots. PAL inactivation was not caused by a soluble inhibitor. Our results indicate that VA fungi initiate a host defense response in alfalfa roots, which is subsequently suppressed.

Salicylic Acid induces cyanide-resistant respiration in tobacco cell-suspension cultures.

Cyanide-resistant, alternative respiration in Nicotiana tabacum L. cv Xanthi-nc was analyzed in liquid suspension cultures using O(2) uptake and calorimetric measurements. In young cultures (4-8 d after transfer), cyanide inhibited O(2) uptake by up to 40% as compared to controls. Application of 20 mum salicylic acid (SA) to young cells increased cyanide-resistant O(2) uptake within 2 h. Development of KCN resistance did not affect total O(2) uptake, but was accompanied by a 60% increase in the rate of heat evolution from cells as measured by calorimetry. This stimulation of heat evolution by SA was not significantly affected by 1 mm cyanide, but was reduced by 10 mm salicylhydroxamic acid (SHAM), an inhibitor of cyanide-resistant respiration. Treatment of SA-induced or uninduced cells with a combination of cyanide and SHAM blocked most of the O(2) consumption and heat evolution. Fifty percent of the applied SA was taken up within 10 min, with most of the intracellular SA metabolized in 2 h. 2,6-Dihydroxybenzoic and 4-hydroxybenzoic acids also induced cyanide-resistant respiration. These data indicate that in tobacco cell-suspension culture, SA induces the activity and the capacity of cyanide-resistant respiration without affecting the capacity of the cytochrome c respiration pathway.

Development and Partial Characterization of Nearly Isogenic Pea Lines (Pisum sativum L.) that Alter Uptake Hydrogenase Activity in Symbiotic Rhizobium.

Some Rhizobium bacteria have H(2)-uptake (Hup) systems that oxidize H(2) evolved from nitrogenase in leguminous root nodules. Pea (Pisum sativum L.) cultivars ;JI1205' and ;Alaska' produce high Hup (Hup(++)) and moderate Hup (Hup(+)) phenotypes, respectively, in Rhizobium leguminosarum 128C53. The physiological significance and biochemical basis of this host plant genetic effect are unknown. The purpose of this investigation was to advance basic Hup studies by developing nearly isogenic lines of peas that alter Hup phenotypes in R. leguminosarum strains containing hup genes. Eight pairs of nearly isogenic pea lines that produce Hup(++) and Hup(+) phenotypes in R. leguminosarum 128C53 were identified in 173 F(2)-derived F(6) families produced from crosses between JI1205 and Alaska. Tests with the pea isolines and three strains of hup-containing R. leguminosarum showed that the isolines altered Hup activity significantly (P

Flavone limitations to root nodulation and symbiotic nitrogen fixation in alfalfa.

Transcription of the nodABC genes in Rhizobium meliloti is required for root nodule formation in alfalfa (Medicago sativa L.) and occurs when specific compounds, such as the flavone luteolin, are supplied by the host plant. Results reported here indicate how luteolin in the root and rhizosphere can affect subsequent N(2) fixation and plant growth. Previous experiments with ;Hairy Peruvian 32' (HP32), an alfalfa population produced from ;Hairy Peruvian' (HP) by two generations of selection for increased N(2) fixation and growth, found that HP32 had more root nodules and fixed more N(2) than the parental HP population. In the present study, flavonoid extracts of HP32 seedling roots are shown to contain a 60% higher concentration of compounds that induce transcription of a nodABC-lacZ fusion in R. meliloti than comparable extracts of HP roots. Chromatographic data indicated that HP32 roots had a 77% higher concentration of luteolin than HP roots. Adding 10 micromolar luteolin to the rhizosphere of HP seedlings increased nodulation, N(2) fixation, total N, and total dry weight but had no effect on nitrate assimilation. These data show that normal levels of flavone nodulation signals in the rhizosphere of HP alfalfa can limit root nodulation, symbiotic N(2) fixation, and seedling growth and suggest that one mechanism for increasing N(2) fixation can be the genetic enhancement of specific biochemical signals which induce nodulation genes in Rhizobium.

Distribution of hydrogen-metabolizing bacteria in alfalfa field soil.

H(2) evolved by alfalfa root nodules during the process of N(2) fixation may be an important factor influencing the distribution of soil bacteria. To test this hypothesis under field conditions, over 700 bacterial isolates were obtained from fallow soil or from the 3-mm layer of soil surrounding alfalfa (Medicago sativa L.) root nodules, alfalfa roots, or bindweed (Convolvulus arvensis L.) roots. Bacteria were isolated under either aerobic or microaerophilic conditions and were tested for their capacity to metabolize H(2). Isolates showing net H(2) uptake and H(2) incorporation activity under laboratory conditions were assigned a Hup phenotype, whereas organisms with significant H(2) output capacity were designated as a Hout phenotype. Under aerobic isolation conditions two Hup isolates were obtained, whereas under microaerophilic conditions five Hup and two Hout isolates were found. The nine isolates differed on the basis of 24 standard bacteriological characteristics or fatty acid composition. Five of the nine organisms were isolated from soil around root nodules, whereas the other four were found distributed among the other three soil environments. On the basis of the microaerophilic isolations, 4.8% of the total procaryotic isolates from soil around root nodules were capable of oxidizing H(2), and 1.2% could produce H(2). Two of the Hup isolates were identified as Rhizobium meliloti by root nodulation tests, but the fact that none of the isolates reduced C(2)H(2) under the assay conditions suggested that the H(2) metabolism traits were associated with various hydrogenase systems rather than with nitrogenase activity. Results from this study support the concept that H(2) evolution by alfalfa root nodules has a significant effect on the surrounding microenvironment and influences the number and diversity of bacteria occupying that region.