Plant Defense Signaling: How is this related to good medicine?

Botrytis cinerea growing on a PDA
Botrytis cinerea growing on a PDA (Wikipedia)

Plants make numerous small molecules (metabolites) that are either directly toxic to insects, grazing animals, fungi and bacteria, or that stimulate the production of other toxic metabolites. The production of various types of metabolites such as alkaloids, terpenes, and phenolics can be turned on (induced) after damage to the plant occurs. The control mechanisms involve  jasmonate, salicylate, phytohormone (plant hormone) ethylene, the volatile (gaseous) methyljasmonate and methylsalicylate signaling pathways.

The signal process is complex. It works on a local level or systemically by traveling throughout the entire plant. It behaves like a network, allowing chemical communication between parts of a plant or within a population of plants in a given locale. With recent developments in metabolic profiling, highly sensitive separation and detection systems are used to create metabolite profiles, high-throughput gene expression analysis is used to detect genes transcripts and rigorous statistical mining resulted in some interesting data that reveals more about how plant defense signaling is controlled.

Researchers used metabolic profiling of overall patterns rather than relying on targeted metabolites. This is important, since previous work focused on a few major metabolites and provided conflicting data. They found differences in profiles from plants exposed to generalist insect feeders versus plants treated with phytohormones. Although both jasmonate and salicylate pathways were activated in each treatment (co-induced), the metabolite patterns were distinct; both treatments lead to a stronger localized rather than systemic response; and there appeared to be a great deal of cross-talk between both pathways influencing pools of precursor metabolites.

Botrytis cinerea growing on tomato leaf
Botrytis cinerea growing on tomato leaf

Another study followed the spatial accumulation of hydroxycinnamates (phenylpropanoids) and lignins in cell walls of Arabidopsis (a model organism) in response to changes in the ethylene signaling induced by the necrotrophic fungal pathogen, Botrytis cinerea. They correlated metabolic profiles with cytological (cell based) changes to provide biological validation of the analytical data.

When a fungus like Botrytis attacks a plant, it generally destroys cells and eventually the entire plant. In the presence of the fungus, plant genes for the biosynthesis of phenylpropanoids and lignins are expressed (turned on) to modify and reinforce the plant cell wall against fungal penetration. Botrytis induces over 30 ethylene regulated transcription factors – cellular molecules that target and induce underlying genes to become active. So these researchers used a metabolite profile of 3 ethylene mutants, plants that had gene mutations at different DNA sequence points of the ethylene signaling pathway.

It turned out that the mutant plants were less resistant to the fungus and that ethylene resistance, when present, appeared after the fungus had made contact with the plant cell wall and had begun to build the structures necessary to penetrate the cell wall barrier. One phenylpropanoid metabolite in particular, ferulate, seemed to be highly influenced by ethylene signaling. Ferulate cross-links the polymer strands of cell wall polysaccharides, enhancing their structural integrity as a barrier.

So what does this have to do with good medicine? Don’t focus on one or two metabolites to make an efficacious extract. Despite what you hear, we are not trying to mimic “magic bullet” medicine. Expose plants to a full set of ecological challenges to produce a metabolite profile of greater diversity. Mono-crop farming doesn’t cut it. Damn if those hippies had it right after all.

Using ePortfolios to Guide Student Learning, Part I

This post also focuses on complex ecologies, found in education, not the wild. It reviews the adoption of an ePortfolio in our Therapeutic Herbalism Masters program at Maryland University of Integrative Health, which was designed to accomplish three main tasks:

  1. Encourage and provide opportunities for our students to experience meta-cognitive learning about the competencies they have acquired/developed..
  2. Create a professional web presence for the student to market their expertise.
  3. Assess whether our program learning outcomes are being met in the classroom.

The Student Learning Portfolio allowed student to collect artifacts of their acquired competencies. These were often based on assignments from each of their courses.  Ideally, they reflected skills developed, competencies, and career readiness resulting from each course and the integration of those experiences. The process was meant to enhance the ability of students to be self-sufficient reviewing their ability to succeed in a chosen profession.

Image of learning Zones

Their final product was creation of a Professional ePortfolio, where the competency artifacts selected by a student showcased the knowledge, skills, and abilities that are in demand in the professional marketplace.

During our review of the tool it became apparent two important lynch pins were missing. Firstly was the articulation of appropriate professional competencies as measurable program learning outcomes. Secondly, our faculty had limited experience applying learning outcomes and reviewing meta-cognitive tools such as the ePortfolio. They were not prepared to highlight the connection between chosen artifact from their course, as well as the underlying learning process and how it was linked to an overriding professional competency.

A series of faculty retreats refined more effective and measurable program learning outcomes. In addition, the institutional assessment process that emerged out of a regular Middle States Higher Education Commission review of the university helped create more specific measurement goals. The combined effect enabled both faculty and students to identify appropriate artifacts and learning processes.

We articulated specific competencies that were missing or ill-defined in previous versions, including:

  • Improved research literacy skills – finding and assessing the validity of scientific research – in support of their analytical work on assignments.
  • Succinct summarization of primary, peer-reviewed resources and the synthesizing of new ideas from those summaries that contribute unique ideas to the field
  • For clinical students, populated their portfolio with case study write-ups.
  • Created effective narratives of how they worked with incomplete data (medicine making, research or diagnosis) in finding a solution using iterative problem solving.

Other challenges that appeared included the need to help students learn how to select learning artifacts that reflect project-based learning. Since the feedback loop in assessment provides data about how program outcomes are being met in the classroom, the process of student metacognitive review of both object and learning processes revealed difficulty in effectively linking the two.

Apparently this is not unusual in ePortfolio development (Land & Greene, 2000). At the core of this issue is the requirement for more engaged and knowledgeable faculty to embed assignments and course long assessment arcs focused on strengthening the linkage between an object (paper, case study, etc.…) and the underlying programmatic learning objective.

Faculty training in applying ePortfolio to their own professional development would improve their the ability to guide students into choosing suitable learning artifact and how to articulate those to employment marketplace.

Reference

Land, S.M. & Greene, B.A. (2000) Project-based learning with the world wide web: A qualitative study of resource integration. Educational Technology Research and Development. 48: 45. https://doi.org/10.1007/BF02313485.

Herding Cats

Image of Cat
Empress Mow Mow

At times during the debate around invasive species, it’s the absurd that strikes the strongest chord. Prevention is a reasonable approach. Eradication after full-scale establishment seems like folly. Evolutionarily, it’s a whole system problem. Take the example invasive Fire Ants (Solenopsis invicta) that originated in Mato Grosso, Brazil. If you’ve ever been bitten, you know they sting. They first appeared in the United States at Mobile, Alabama around 1940. How they were introduced is unknown.

Real, potential solutions the control of their populations appear to be phorid flies that lay their eggs inside the ant’s head and fungi that are natural predators/parasites of the ant in their original ecosystem. Go back to source, investigate complex ecologies for solutions. But think through the impact on the new system you are invading.

Most recently humans have been a major cause of jump dispersal, another name for invasive species. But hasn’t much of evolutionary spread of living organisms been the product of “invasive/exotic” species from the very beginning?

Image of Dog
Trip Gibson

So, if we bring on the cats, then don’t we need more dogs?

Everybody’s Dancing with Every One Else

A paper out of the Hawes Lab noted that root border cells respond to or probe the surrounding soil by releasing a multitude of proteins, amino acids, sugars and secondary metabolites, and they do so even after the cells detach from the root cap. This material, called exudates, appears to alter the surrounding rhizosphere community of microorganismsCurlango-Rivera et al., (2010) investigated the consequence of these plant exudates on the growth and production of root border cells. Exposure to pisatin, at concentrations capable of inhibiting fungal growth, stimulated production of root border cells, while exposure to a plant cell wall component, ferulic acid, inhibited growth of root border cells. Pisatin is a  phytoalexin, a secondary metabolite made by the plant to limit microbial attack.

Ferulic Acid
Ferulic Acid

Does this indicate that plant root investment in root border cells as sensory organs occurs under the protective influence of a plant phytoalexins, but that cell wall fragments resulting from the rupture of root cap cells under microbial attack may limit plant root border cell proliferation? I am curious about experimental results if simultaneous exposure to the compounds occurred? Ferulic acid is often bound to a matrix of polymer structures found in plant cell walls. Would it take time for diffusion of these compounds into the rhizosphere to gain concentrations able to impart a biological effect?

Fermentation as an Extraction Method

Although most cultures have fermented food as a staple in their dietary patterns, little has been reported on the use of fermentation as an herbal extraction method. The process may contribute more than just modifying solvent pH. Rizzello et al. (2013) reported using a lactic acid fermentation with specific yeast strains that improved the antioxidant activity of Echinacea purpurea. They compared fermentation extraction to either a methanol or water extract without fermentation. The greatest antimicrobial activity was associated with low molecular mass compounds negated in the presence of digestive enzymes, suggesting small peptides as the active agent.  The authors cited other experiments with grapes, soy and cereal grains where the fermentation process increased bio-availability of  certain compounds and produced novel chemical species.

This process is worth exploring by both herbal supplement companies and herbalist as a new medicine making method. Traditional texts provide some guidance.  Enzymatic processes can optimize extraction of plant cellular content at lower temperatures. And research (Mishra et al., 2010; Mulay and Khale, 2011) applying traditional Ayurvedic methods of  fermented extraction found reduced toxicity in the final product. This opens up a little explored market around functional foods as well.

Reference:

  1. Mishra AKGupta AGupta VSand RBansal P2010Asava and arishta: an Ayrvedic medicine – an overview. Int J Pharm Biol Arch. 1(1):2430.
  2. Mulay SKhale A2011Asavarishtas through improved fermentation technology. Int J Pharma Sci Res. 2(6):14211425.
  3. Rizzello, CG et al. (2013) Lactic acid fermentation as a tool to enhance the functional features of Echinacea spp. Microbial Cell Factories. 12:44-59.

 

Not Blueberry Pie, but Close

Vaccinium myrtillus
Image via Wikipedia

Bilberry (Vaccinium myrtillus) contains varying levels of phenolic compounds – anthocyanins, chlorogenic acid derivatives, hydroxycinnamic acids, flavonol glycosides, catechins, and proanthocyanidins. Research by Martz et al (2010) elucidated how levels of bilberry leaf phenolics differed along an ecological gradient in boreal forests running north to south in Finland. These regions differ in latitude, altitude, over story cover, levels of continuous light, temperature and associated frost spells.

An analysis of bilberry leaves showed that major phenolic changes in bilberry leaves appeared in the first stages of leaf development. As important, synthesis and accumulation of flavonoids was delayed in the forest compared to the high light sites. Two-fold higher flavonoid levels appeared in leaf tissue growing in high-light intensity sites, higher latitudes, and/or higher altitudes compared to in lower altitudes and low-light intensity sites.

Close and Mcarther (2002) previously theorized that the presence of greater phenolic levels in leaf tissue found in northern regions was a response to colder temperatures, which would limit essential enzyme function, during periods of maximal photo-oxidative stress (Close and Mcarther, 2002).  However, Martz et al (2010) also showed that leaf flavanoid genes were highly expressed in shade, but that the timing of expression appeared to alter the relative metabolite levels in shade compared to sun exposed bilberry leaf.

Mudge et al., (2016), researched phenolic profiles of wild elderberry fruits (Sambucus nigra subsp. canadensis) over two years in eastern US, noting that flavanols (quercitin, isoquercitin, rutin) and chlorogenic acid metabolite concentrations were higher in the southeast, particularly interior. They suggested the variation of phytochemical profiles of the berries were impacted by genetic or environmental factors without understanding on which was more important.

What’s missing from the data picture includes a more complex measurement of ecological influences, such as response to herbivory and rhizosphere fungal associations? This type of whole community data would help to build a more complete picture of  plant response.

This requires sampling, sampling, sampling.

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  1. Martz, F., Jaakola, L., Julkunen-Tiitto, R. and Stark, S. (2010). Phenolic Composition and Antioxidant Capacity of Bilberry (Vaccinium myrtillus) Leaves in Northern Europe Following Foliar Development and Along Environmental Gradients. J Chem Ecol, published online, 19 August 2010
  2. Close, D.C., and Mcarther, C. (2002). Rethinking the role of many plant phenolics—protection from photodamage not herbivores? Oikos. 99:166–172
  3. Mudge, E., Applequist, W. L., Finley, J., Lister, P., Townesmith, A. K., Walker, K. M., & Brown, P. N. (2016). Variation of Select Flavonols and Chlorogenic Acid Content of Elderberry Collected Throughout the Eastern United States. Journal of food composition and analysis : an official publication of the United Nations University, International Network of Food Data Systems, 47, 52–59.

Ginseng Allelopathy in the Rhizosphere

The active, therapeutic phytochemicals in the Panax spp. appear to be ginsenosides that consist of an aglycone base structure and glycosides (sugar molecules). These compounds can be referred to as triterpene glycosides, triterpene saponins and steroid saponins. The varied nomenclature comes from the multiple ways of defining the molecule. Saponins froth when shaken. And from the image below, ginsenoside aglycones contain a steroid backbone. Based on the numbers of carbons they can also be classified as a triterpene. 

Ginsensodie Steroid

So what role do these phytochemicals they play on the plants behalf? Saponins as class have anti-fungal properties and may act allelopathically (Carter et al., 1999). The sterol portion of the molecule appears to be inserted into and disrupt fungal membrane integrity by interacting with fungal sterols present.

Nicole et al. (2002) noted that American ginseng saponins inhibit in vitro growth of the fungus Trichoderma spp., but stimulate growth of Cylindrocarpon destructans.

In a followup study to  investigate the concentration of ginsensosides in rhizosphere, Nicol et al. (2003) collected ginsenosides from root associated soil several times between 1999-2002. They found that the concentration in the soil ranged 0.02 – 0.098%. They also collected root exudate from pot-grown ginseng over 22 days, using an exudate trapping system,  which yielded a concentration of 0.6% ginsenosides. We need to test whether this soil concentration level can be considered an active allelopathic level.

Additional evidence supporting the sterol disruption hypothesis can be found in the Pythiaceous fungi (especially Pythium spp. and Phytophthora spp.) that lack sterols in their  cell walls.  Growth of these fungi appear to be stimulated by both the presence of fungal sterols (ergosterol) and ginsenosides in the medium of in vitro studies. The authors suggested potential mechanisms include: 

The ginsenosides…

  • provide a carbon sink for the fungus.
  • alter fungal membranes in a positive manner.
  • act as a fungal growth hormone.

Future studies should look at the ratio of ginsensosides and their respective influence on fungi.

References:

  1. Carter, JP, Spink, J, Cannon, PF, Daniels, and Osbourn, AE. (1999) Isolation, Characterization, and Avenacin Sensitivity of a Diverse Collection of Cereal-Root- Colonizing Fungi.AppliedandEnvironmentalMicrobiology. 65(8): 3364–3372.
  2. Nicol, RW, Traquair, JA and , Bernards, MA. (2002) Ginsenosides as host resistance factors in American ginseng (Panaxquinquefolius).CanadianJournalofBotany. 80(5): 557-562.
  3. Nicol RW, Yousef L, Traquair JA, and Bernards MA. (2003) Ginsenosides stimulate the growth of soilborne pathogens of American ginseng.Phytochemistry. 64(1):257-64.