Punicalagin, a pomegranate polyphenol sensitizes the activity of antibiotics against three MDR pathogens of the Enterobacteriaceae.

BACKGROUND: Multidrug resistance (MDR) in the family Enterobacteriaceae is a perniciously increasing threat to global health security. The discovery of new antimicrobials having the reversing drug resistance potential may contribute to augment and revive the antibiotic arsenal in hand. This study aimed to explore the anti-Enterobacteriaceae capability of bioactive polyphenols from Punica granatum (P. granatum) and their co-action with antibiotics against clinical isolates of Enterobacteriaceae predominantly prevalent in South Asian countries. METHODS: The Kandhari P. granatum (Pakistani origin) extracts were tested for anti-Enterobacteriaceae activity by agar well diffusion assay against MDR Salmonella enterica serovar Typhi, serovar Typhimurium and Escherichia coli. Predominant compounds of active extract were determined by mass spectrometry and screened for bioactivity by agar well diffusion and minimum inhibitory concentration (MIC) assay. The active punicalagin was further evaluated at sub-inhibitory concentrations (SICs) for coactivity with nine conventional antimicrobials using a disc diffusion assay followed by time-kill experiments that proceeded with SICs of punicalagin and antimicrobials. RESULTS: Among all P. granatum crude extracts, pomegranate peel methanol extract showed the largest inhibition zones of 25, 22 and 19 mm, and the MICs as 3.9, 7.8 and 7.8 mg/mL for S. typhi, S. typhimurium and E. coli, respectively. Punicalagin and ellagic acid were determined as predominant compounds by mass spectrometry. In plate assay, punicalagin (10 mg/mL) was active with hazy inhibition zones of 17, 14, and 13 mm against S. typhi, S. typhimurium and E. coli, respectively. However, in broth dilution assay punicalagin showed no MIC up to 10 mg/mL. The SICs 30 µg, 100 µg, and 500 µg of punicalagin combined with antimicrobials i.e., aminoglycoside, ß-lactam, and fluoroquinolone act in synergy against MDR strains with % increase in inhibition zone values varying from 3.4 ± 2.7% to 73.8 ± 8.4%. In time-kill curves, a significant decrease in cell density was observed with the SICs of antimicrobials/punicalagin (0.03-60 µg/mL/30, 100, 500 µg/mL of punicalagin) combinations. CONCLUSIONS: The P. granatum peel methanol extract exhibited antimicrobial activity against MDR Enterobacteriaceae pathogens. Punicalagin, the bacteriostatic flavonoid act as a concentration-dependent sensitizing agent for antimicrobials against Enterobacteriaceae. Our findings for the therapeutic punicalagin-antimicrobial combination prompt further evaluation of punicalagin as a potent activator for drugs, which otherwise remain less or inactive against MDR strains.

Quantification of plant cardenolides by HPLC, measurement of Na+/K+-ATPase inhibition activity, and characterization of target enzymes.

The biosynthesis of cardiac glycosides, broadly classified as cardenolides and bufadienolides, has evolved repeatedly among flowering plants. Individual species can produce dozens or even hundreds of structurally distinct cardiac glycosides. Although all cardiac glycosides exhibit biological activity by inhibiting the function of the essential Na+/K+-ATPase in animal cells, they differ in their level of inhibitory activity. For within- and between-species comparisons of cardiac glycosides to address ecological and evolutionary questions, it is necessary to not only quantify their relative abundance, but also their effectiveness in inhibiting the activity of different animal Na+/K+-ATPases. Here we describe protocols for characterizing the amount and toxicity of cardenolides from plant samples and the degree of insect Na+/K+-ATPase tolerance to inhibition: (1) an HPLC-based assay to quantify the abundance of individual cardenolides in plant extracts, (2) an assay to quantify inhibition of Na+/K+-ATPase activity by plant extracts, and (3) extraction of insect Na+/K+-ATPases for inhibition assays.

Tecia solanivora infestation increases tuber starch accumulation in Pastusa Suprema potatoes.

In response to infestation with larvae of the Guatemalan tuber moth (Tecia solanivora), some Solanum tuberosum (potato) varieties exhibit an overcompensation response, whereby the total dry mass of uninfested tubers is increased. Here, we describe early responses, within the first few days, of T. solanivora feeding, in the Colombian potato variety Pastusa Suprema. Non-targeted metabolite profiling showed significant secondary metabolism changes in T. solanivora-infested tubers, but not in uninfested systemic tubers. In contrast, changes in primary metabolism were greater in uninfested systemic tubers than in the infested tubers, with a notable 80% decline in systemic tuber sucrose levels within 1 d of T. solanivora infestation. This suggested either decreased sucrose transport from the leaves or increased sink strength, i.e., more rapid sucrose to starch conversion in the tubers. Increased sucrose synthesis was indicated by higher rubisco activase and lower starch synthase gene expression in the leaves of infested plants. Elevated sink strength was demonstrated by 45% more total starch deposition in systemic tubers of T. solanivora-infested plants compared to uninfested control plants. Thus, rather than investing in increased defense of uninfested tubers, Pastusa Suprema promotes deposition of photoassimilates in the form of starch as a response to T. solanivora infestation.

Concurrent Overexpression of Arabidopsis thaliana Cystathionine γ-Synthase and Silencing of Endogenous Methionine γ-Lyase Enhance Tuber Methionine Content in Solanum tuberosum.

Potatoes (Solanum tuberosum) are deficient in methionine, an essential amino acid in human and animal diets. Higher methionine levels increase the nutritional quality and promote the typically pleasant aroma associated with baked and fried potatoes. Several attempts have been made to elevate tuber methionine levels by genetic engineering of methionine biosynthesis and catabolism. Overexpressing Arabidopsis thaliana cystathionine γ-synthase (AtCGS) in S. tuberosum up-regulates a rate-limiting step of methionine biosynthesis and increases tuber methionine levels. Alternatively, silencing S. tuberosum methionine γ-lyase (StMGL), which causes decreased degradation of methionine into 2-ketobutyrate, also increases methionine levels. Concurrently enhancing biosynthesis and reducing degradation were predicted to provide further increases in tuber methionine content. Here we report that S. tuberosum cv. Désirée plants with AtCGS overexpression and StMGL silenced by RNA interference are morphologically normal and accumulate higher free methionine levels than either single-transgenic line.

RNAi-Mediated Simultaneous Resistance Against Three RNA Viruses in Potato.

RNA interference (RNAi) technology has been successfully applied in stacking resistance against viruses in numerous crop plants. During RNAi, the production of small interfering RNAs (siRNAs) from template double-standard RNA (dsRNA) derived from expression constructs provides an on-switch for triggering homology-based targeting of cognate viral transcripts, hence generating a pre-programmed immunity in transgenic plants prior to virus infection. In the current study, transgenic potato lines (Solanum tuberosum cv. Desiree) were generated, expressing fused viral coat protein coding sequences from Potato virus X (PVX), Potato virus Y (PVY), and Potato virus S (PVS) as a 600-bp inverted repeat expressed from a constitutive 35S promoter. The expression cassette (designated Ec1/p5941) was designed to generate dsRNAs having a hairpin loop configuration. The transgene insertions were confirmed by glufosinate resistance, gene-specific PCR, and Southern blotting. Regenerated lines were further assayed for resistance to virus inoculation for up to two consecutive crop seasons. Nearly 100% resistance against PVX, PVY, and PVS infection was observed in transgenic lines when compared with untransformed controls, which developed severe viral disease symptoms. These results establish the efficacy of RNAi using the coat protein gene as a potential target for the successful induction of stable antiviral immunity in potatoes.

Costs and Tradeoffs of Resistance and Tolerance to Belowground Herbivory in Potato.

The success of sustainable crop production depends on our ability to select or create varieties that can allocate resources to both growth and defence. However, breeding efforts have emphasized increases in yields but have partially neglected defence traits against pests. Estimating the costs of multiple defences against tuber herbivores and the tradeoffs among them, as well as understanding the relationship between yield and multiple defences is still unknown but relevant to both basic and applied ecology. Using twenty commercial potato varieties available in Colombia and the tuber herbivore Tecia solanivora, we tested whether high yielding varieties show a reduction in three types of defence: constitutive and induced resistance, as well as tolerance. Specifically, we determined (1) the costs in terms of yield of all three defences, (2) the possible tradeoffs among them, and (3) if oviposition preference was related to the expression of these defences. We detected no costs in terms of yield of constitutive and induced resistance to tuber damage. We did, however, find evidence of costs of being able to tolerate tuber herbivory. While we found no tradeoffs among any of the estimated defences, there was a positive correlation between aboveground compensatory growth and tolerance in terms of tuber production, suggesting that after damage there are no shifts in the allocation of resources from aboveground to belowground biomass. Finally, we found that females laid more eggs on those varieties with the lowest level of constitutive resistance. In conclusion our findings suggest that in potatoes, breeding for higher yields has not caused any reduction in constitutive or induced resistance to tuber damage. This is not the case for tolerance where those varieties with higher yields are also less likely to tolerate tuber damage. Given the high incidence of tuber pests in Colombia, selecting for higher tolerance could allow for high productivity in the presence of herbivores. Finding mechanisms to decouple the tolerance response from yield should be a new priority in potato breeding in Colombia to guarantee a higher yield in both the presence and absence of herbivores.

Potato tuber herbivory increases resistance to aboveground lepidopteran herbivores.

Plants mediate interactions between aboveground and belowground herbivores. Although effects of root herbivory on foliar herbivores have been documented in several plant species, interactions between tuber-feeding herbivores and foliar herbivores are rarely investigated. We report that localized tuber damage by Tecia solanivora (Guatemalan tuber moth) larvae reduced aboveground Spodoptera exigua (beet armyworm) and Spodoptera frugiperda (fall armyworm) performance on Solanum tuberosum (potato). Conversely, S. exigua leaf damage had no noticeable effect on belowground T. solanivora performance. Tuber infestation by T. solanivora induced systemic plant defenses and elevated resistance to aboveground herbivores. Lipoxygenase 3 (Lox3), which contributes to the synthesis of plant defense signaling molecules, had higher transcript abundance in T. solanivora-infested leaves and tubers than in equivalent control samples. Foliar expression of the hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) and 3-hydroxy-3-methylglutaryl CoA reductase I (HMGR1) genes, which are involved in chlorogenic acid and steroidal glycoalkaloid biosynthesis, respectively, also increased in response to tuber herbivory. Leaf metabolite profiling demonstrated the accumulation of unknown metabolites as well as the known potato defense compounds chlorogenic acid, α-solanine, and α-chaconine. When added to insect diet at concentrations similar to those found in potato leaves, chlorogenic acid, α-solanine, and α-chaconine all reduced S. exigua larval growth. Thus, despite the fact that tubers are a metabolic sink tissue, T. solanivora feeding elicits a systemic signal that induces aboveground resistance against S. exigua and S. frugiperda by increasing foliar abundance of defensive metabolites.

The catabolic enzyme methionine gamma-lyase limits methionine accumulation in potato tubers.

Increasing methionine in potato tubers is desirable, both to increase the availability of this limiting essential amino acid and to enhance the aroma of baked and fried potatoes. Previous attempts to elevate potato methionine content using transgenic approaches have focused on increasing methionine biosynthesis. Higher isoleucine accumulation in these transgenic tubers suggested that the potatoes compensate for increased methionine biosynthesis with enhanced catabolism via methionine gamma-lyase (MGL), thereby producing 2-ketybutyrate for isoleucine biosynthesis. In the current study, we show that potato StMGL1 encodes a functional MGL in potato tubers. In planta silencing of StMGL1 results in an increased methionine to isoleucine ratio in the free amino acid profile of potato tubers and, in some transgenic lines, elevated accumulation of free methionine. In both wild-type and transgenic tubers, the ratio of methionine to isoleucine is negatively correlated with the level of StMGL1 transcript. A three-dimensional distribution of free amino acids in potato tubers is also described.

Grass roots chemistry: meta-tyrosine, an herbicidal nonprotein amino acid.

Fine fescue grasses displace neighboring plants by depositing large quantities of an aqueous phytotoxic root exudate in the soil rhizosphere. Via activity-guided fractionation, we have isolated and identified the nonprotein amino acid m-tyrosine as the major active component. m-Tyrosine is significantly more phytotoxic than its structural isomers o- and p-tyrosine. We show that m-tyrosine exposure results in growth inhibition for a wide range of plant species and propose that the release of this nonprotein amino acid interferes with root development of competing plants. Acid hydrolysis of total root protein from Arabidopsis thaliana showed incorporation of m-tyrosine, suggesting this as a possible mechanism of phytotoxicity. m-Tyrosine inhibition of A. thaliana root growth is counteracted by exogenous addition of protein amino acids, with phenylalanine having the most significant effect. The discovery of m-tyrosine, as well as a further understanding of its mode(s) of action, could lead to the development of biorational approaches to weed control.