Good Wine, Good Fungi

A study of organic soils found that the those associated with organic gardening compared to conventional methods or native grasslands, was very similar in types and diversity of mycorrhizal fungal taxa to that of the native soils. Increasingly, viticulturalists have been promoting the sustainability of using organic techniques over the fungicide heavy approaches of conventional wine management practices, and that this fundamental investment in “terroir” makes better wine. One method is to restore the density and diversity of beneficial, symbiotic fungi in the vineyard soil. These fungi are seriously depleted in soils that have had extensive chemical fertilizers, fungicides or pesticides applied.

Mycorrhizal inoculum applied to new vines plantings and as a dressing to cover crop used to improve nitrogen availability in vineyard soils, associates with the vine roots and  increases both the available levels of organic carbon and the water holding capacity of the surrounding soils. And with healthy vines, and a biological approach to vineyard management in place, the rhizosphere community rich in mycorrhizal fungi can influence the quality of wine produced. 

Gabriele et al. (2016) investigated the effect of mycorrhizal inoculation of various Sangiovese wine grapes. The symbiotic relationships improved the oxidative stability, thus the potential ability of the wine to age, and increased 14 polyphenols compared to un-inoculated plants. The later effect may improve the structure and the flavor profile of the wine.

I’ve asked to join the downstream portion of the research team to investigate the impact of these changes on the consumers experience.

What’s the Best Way to Flirt with Mycorrhizal Fungi?

Signaling molecules from either plant or fungi are perceived by the other using receptors. Many plants monitor their ecosystem for bacteria or fungi using receptor-kinases, which as cell surface proteins activate a signaling cascade in the cell to change it’s function in some way. Research groups continue to unearth various themes on this mechanisms for plant/mycorrhizal communication.

One model, identified Lipochitooligosaccharides (LCO) as signal molecules used by nitrogen fixing bacteria (rhizobia) to alter how plant roots form a symbiotic relationship. Communication using LCOs allows plants to gain nitrogen from soil bacteria and bacteria to gain carbon in the form of plant sugars. Similar molecules are excreted by arbuscular mycorrhizal (AM) fungi. This research noted that a mixture of sulphated and non-sulphated lipochitooligosaccharides (LCOs) secreted from the AM fungi, Glomus intraradices, stimulated root branching and growth in the legume Medicago truncatula. Apparently, the diffusible chemicals activated plant root genes that code for a series of receptor kinase. In M. truncatula, rhizobium LCO secretions also stimulate the same symbiotic pathway. The researchers found this signaling effect active in diverse plant species.

In other experiments, scientists found a hydrolase protein (D14L), which functions deep within the cell, modulating plant communication with AM fungi. This receptor had originally been characterized as a receptor for Karrikin, a plant hormone produced when plant material is burned. In species such as eucalyptus and the tobacco family, this hormone detects smoke and stimulates seed germination after fire has decimated an ecosystem. It allows those plants, known as fire chasers, to outcompete in the newly altered environmentWhat is particularly interesting – the same protein is part of early plant developmental interaction with light, and may have played an evolutionary role in plant emergence on to land.

So burn a little incense, light a candle, offer up something sweet and see if your mycorrhizal fungus responds. You don’t need to burn down the entire house!

Outpost Communication

Dr.  Martha Hawes has been a pioneering researcher on plant root border cells. I became fascinated with their role while researching the fungal/plant communication in the rhizosphere of goldenseal (Hydrastis canadendis) during my doctorate. I called her lab hoping someone might speak with me. She answered and spent an hour pointing out important research papers and suggesting approaches I might take to incorporate root border cell research. She was always open to helping anyone with a curious mind and passion for the subject into which she’d immersed her career efforts. I’m grateful to her for showing me generosity and kindness.

Plant root border cells are formed at the root tip where physical and biological interactions occur with the soil and microbe communities. The cells are genetically programmed to separate from the rest of the root structure and from each other. Cell-wall degrading enzymes dissolve cell wall matrix material that holds plant cells together. These “outpost” remain biologically active, excreting proteins and smaller molecules into the surrounding environment. Both types of molecules act as signals turning on/off gene expression to stimulate or prevent the growth of soil-borne bacteria and fungi. One important role appears to be in establishing a symbiotic relationship with mycorrhizal fungi (see previous post).

Few plants such as the Arabidopsis thaliana, which do not produce root border cells, also do not form mycorrhizal associations. In most plants, the content of border cells are accessible only to microorganisms able to recognize and respond to specific root signals. Among the compounds located in root border cells of various plants, medicinally valuable isoflavonoids modulate stable ecological relationships between mycorrhizal fungi and plant root tissue. These fungi stimulate the production of isoflavonoid in plant root tissue, while simultaneously the isoflavonoids increase mycorrhizal spore germination. The spores are an important survival mechanism used by the fungi. Measuring the activity in root border cells in “real time” as they interact with fungi is one of the great challenges to plant biologists.

Here’s a short video showing the release of border cells from a plant root cap:

More in-depth readings:
Harrison, M. and Dixon, R. (1993) Isoflavonoid accumulation and expression of defense gene transcripts during establishment of vesicular-arbuscular mycorrhizal associations in roots of Medicago truncatula. Mol. Plant Microbe Interact. 6:643-654
Hawes, M,C. et al (1998) Function of root border cells in plant health: Pioneers in the Rhizosphere, Annual Review of Phytopathology, 36:311-327.
Hawes, M.C. et al (2003) Root Caps and Rhizosphere. J. Plant Growth Reg. 21:353.
Kape, R. et al (1992) Legume root metabolites and VA-mycorrhiza development. J. Plant Physiol. 141:54-60.
Phillips D.A. et al (2004) Microbial products trigger amino acid exudation from plant roots. Plant Phys. 136: 2887-2894

The Real Gumbo Recipe

I wanted to follow up requests in response to my post on Spices, and share the gumbo recipe I mentioned. 

It’s my mom’s from her mom, Nannie (Louise P. Curtis)

I’ve used Nannie’s original language

Start with 1 large TBS bacon, ham or sausage drippings in heavy saucepan

Chop 1 pound of okra, 1 large onion, 1 large bell pepper and 5 cloves of garlic

Fry in oil until wilted. Okra will lose stringiness.

Add 1-2 TBS of flour. Cook slowly about 30-45 minutes until okra gets drier.

Add 1 can tomato paste

Add hot seafood water, about 4 cups

Add seafood 1  lb crabmeat and 1 lb peeled shrimp

Add seasonings:

  • 2 drops Tabasco or cayenne pepper
  • salt and pepper to taste
  • a couple of bay leaves
  • chopped celery (1/4 cup)
  • green onions and parsley (optional)

Simmer for about 1 hour over very low fire

Serve over rice

 

Note: I always added olive oil and Louisiana Crystal Hot Sauce and opened a bottle of Jax or two.

 

Plant Root Chemistry is Just an Invitation to Play

Flavonoid Basic Structure

Plants and organisms in the rhizosphere (area of soil surrounding plant roots) are living organisms and part of a complex ecosystem requiring communication skills. Two different classes of compounds are important communicators – flavonoids and strigolactones – both ubiquitous in plants across multiple taxa. Of note, strigolactones were originally detected (Bouwmeester et al.) in plant root exudate stimulating seed germination of parasitic plants (genera Striga and Orobanche). These weedy species parasites host plant roots for nutrients.

Flavonoid basal structures are highly varied, including flavones, flavonols, flavan-3-ols, flavanones, isoflavonoids, isoflavans and pterocarpans. They accumulate at root tips or root cap and makeup a large portion of root exudate. The fact these structures are easily modified and that their biosynthesis can be triggered by a numerous transcription factors points strongly to a role as elicited, signaling compounds. The conversation starters, the deal makers, they patrol the root neighborhood deciding who’s gonna join the party.

Hassan and Mathesius (2012) noted in more technical terms that this localization allows them to influence the rhizosphere environment – increasing the bioavailability of both phosphorous and iron, inducing Rhizobium nod genes (for nitrogen fixation), determining host specificity, and influencing bacterial quorum sensing. They also influence soil  fungi, both parasite and non-parasite, to investigate their environment by stimulating macronidial germination. These are spore structures that allow the fungus to remain in a dormant state until the surrounding soil supports their growth (Ruan et al.). Plants represent nutrient sources – the internal cell structures for parasites, and the substances released from plant root such as carbohydrates, organic acids, and proteins (root exudate) for non-parasitic fungi.

One of the most fascinating chemical conversations involves how both flavonoids and strigolactones trigger AM fungi to investigate the rhizosphere more actively, by stimulating sporulation (breaking out of their dormant state), hyphal branching (similar to send out runners), and root colonization. Interestingly, changes to flavonoid ratios in root exudate can alter the symbiotic relationship and defines how mature the relationship is developmentally.

Metabolic pathways in living organisms require dedicated gene expression. They tend to have been around a long time. Plants originated as aquatic organisms. They had no root systems. The prevalent theory on their terrestrial adaptation suggests that root exudate facilitated symbiosis with AM fungi, which allowed primitive land plants to survive by providing them an early “root system”.

A paper by Delaux et. al. (2012) tested whether the presence of strigolactones in the aquatic green algae lineage may have helped them adapt to and colonize terrestrial environments. The researchers used a bioassay to detect branching of a AM fungus, Gigaspora rosea, to show strigolactones were present in the green algae Charales corallina. They also applied a synthetic strigolactone to C. coralina, which stimulated rhizoid elongation in the algae. Rhizoids were early “root-like” structures. The results beg the question of whether strigolactone biosynthesis predates AM fungal colonization and reinforces the idea that what survives adapts to changes in habitat, since anchoring to land increased the plant’s ability to acquire water and nutrients.

Strigolactones General Chemical Structure

Experiments on potato found that  strigolactone may be involved in resource partitioning by maintaining phosphate and nitrogen homeostasis in plants (Pasare et al., 2013).  Researchers reported that strigolactones enhanced plant association with AM fungi by increased branching of the AM hyphae (Giovannetti et al.). We see the effect of this type of chemical conversation, both internally within the plant and externally, with the fungus, to stimulate exploration.

Strigolactones do also play a role as a plant hormone and appear to regulate axillary growth, lateral branching, and decreased apical dominance (Delavault et al.). This mimics the biological impact on fungi and suggestions that plants explore their aerial environment of air and sunlight.

Rasmussen et al. (2012) noted the ability of strigolactones to impact root exploration in plants, limiting adventitious rooting by inhibiting the initial formative divisions of founder cells. These phenomena may point to the plant directing both the timing and directionality of new root growth in response to the presence/absence of appropriate soil symbionts.

So then, what triggers strigolactone or flavonid secretions? What’s the chemical cross talk originating from the fungal side of the conversation?

 

 

 

 

 

 

 

 

 

 

Spices

I was lucky enough to grow up all over the world – Thailand, Hong Kong, Germany, Romania and Greece. Some of my fondest memories are around food. My mom was a fabulous cook, which was a vital part of my father’s work as a cultural affairs officer in the US Foreign Service.

Vanilla bean plant

One of my earliest memories was of making a landing in Anchorage Alaska on a return trip from Bangkok to my parents’ home town of Biloxi, Mississippi. I remember the large stuffed polar bear in the lobby, and more importantly, my first taste of vanilla ice cream. Having come from the heat of Bangkok, with my daily food rich in hot peppers and ginger, this cooling fragrant treat was nectar from the gods.

When I started to learn to cook standing over my mom’s pot of gumbo she stressed the importance of simmer with the last ingredients – two bay leaves. They looked so simple – small dried plant material –  but an hour later, the aroma of my Nannie’s gumbo was bigger than just the file and seafood, with the bay leaves providing a vibrant top note.  Years later, deep into a career researching active molecules in medicinal plants, I realized that the food culture I grew up in was full of medicinal plants in the spices added to our evening meals.

Bay Leaf

Spices both sense of taste and smell. Our sense of smell, in particular, is influenced by the characteristics of the molecules responsible for odor. How quickly do they volatilize (how easily molecules vaporize)? How soluble are they in water or oil?  How acidic is the food (a tomato based food or something with lemon)? Even our genes can influence the sense of smell.

A molecule that we can sense through smell is called a fragrance or odorant. Those molecules need to reach the olfactory system in the upper part of our nose. To do so, generally the molecular weight of these molecules needs to be equal to or less than 300 g/mol (a scientific designation for the mass of a substance divided by the amount of the substance). Ultimately, our sense of smell comes down to a pattern of activity of neurons in the brain responding to the stimulus of odor molecules binding to receptors (Malnic et al., 1999).

We have receptors (a protein in a cell membrane that responds to molecule attaching to it) in our nasal passages that are triggered by these molecules. Each olfactory receptor recognizes more than one odorant, and each odorant can be detected by several different olfactory receptors. This reflects a combinatorial process common to biological systems. Several factors influence the resulting signal sent from the receptor site.

We know that the shape of a molecule is important (Saberi and Seyed-Allaei, 2016), and that as the molecule binds (or sticks to) a receptor, the receptor changes shape, which leads to neural signals reaching the brain.  Several theories exist to help us understand how to map the process of detecting odors, based largely on chemical qualities of the odorants.

Mori et al. (1994) suggested that the odor signal are more complex than a single receptor binding event, rather the signal is a product of a series of receptor excitations from numerous receptor sub sites (odotypes). This became known as the Odotope Theory. This theory also explains odor-less molecules as the presence sub sites more numerous than the limit of potential binding sites.

A second important theory, Dyson (1938) originated the Vibrational Theory that Wright (1982) later refined. This theory posits that olfactory receptors might really sense vibrational energy on a quantum level rather than structural shapes of the molecule when detecting odors. Quantum mechanics describes nature at the smallest scales of energy levels of atoms or substances.

Combining the two theories, Turin and Yoshi suggest that the tightness of receptor binding, based on both the physical and charge shape (polarity) of an odorant molecule may influence the intensity of the odor, while the character of the odor is effected by vibrational characteristics of the molecule.

To help visualize the theories, we can take a series of molecules found in medicinal plants that have a common base structure, and explore how their odor characteristics may be influenced by chemical features such as solubility, volatility, molecular shape and size (see Table 1).

Starting with the basic structure, vanillin give vanilla its signature sweet, perfumed, woody aroma. The molecular weight is relatively low, and it volatilizes easily, filling a room with the odor when cooked. The oxygen R-groups (groups that “hang” from the ring) on the benzene ring of vanillin make it highly solubility in water.

Built from the same basic structure, eugenol has a short hydro-carbon tail that gives it a stronger odor than vanillin, the familiar aroma found in bay leaf or allspice. This hydro-carbon tail also makes it more fat soluble and may influence the intensity of receptor binding. The odor threshold of eugenol is also lower than that of vanillin. The less polar binding site on the molecule may influence the strength of binding in this example, and explain why the vanillin odor is not as persistent as that of bay leaf or allspice.

Table 1: Vanillin Based Molecular Structures with Odor Thresholds

Vanillin

(Odor Threshold: 20-200ppb)*

Eugenol

(Odor Threshold: 6-30ppb)

Zingerone

(Odor Threshold: na)

Capsaicin

(Odor Threshold: na)

*http://www.leffingwell.com/odorthre.htm

Ginger plant

Zingerone has an even longer hydro-carbon tail connected to the basic vanillin shape, making it insoluble in water. Found in ginger and mustard oil, it gives a rich, sweet, warm and woody fragrance. However, the presence of the carbonyl group (C=O) in the tail means that the zingerone molecules tend to attract each other, limiting how easily it volatilizes. Thus, ginger is less likely to fill a room as quickly with its aroma than vanilla.

Even though Capsaicin also has a long, hyrdo-carbon rich tail, the polar amide group (-NHCO-) makes it slightly more soluble in water than zingerone. The size of the tail also limits the molecule’s volatility. Capsaicin has no odor, and given the numerous sites along that long tail with the potential of binding to a receptor sub sites, this may be an example of a molecule with too many binding sites, as the Odotope theory suggests.

Chili peppers

So the next time in you are in the kitchen and the aromas are filling your senses, see if you can think about the shapes and characteristics of the molecules influencing that wonderful moment.

 

References:

  1. Malnic B, Hirono J, Sato T, Buck LB. (1999) Combinatorial receptor codes for odors. 96(5), 713.
  2. Saberi M, Seyed-Allaei H (April 2016). “Odorant receptors of Drosophila are sensitive to the molecular volume of odorants”. Scientific Reports. 6: 25103. doi:1038/srep25103.
  3. Mori, K. and Shepherd, GM. (1994). Emerging principles of molecular signal processing by mitral/tufted cells in the olfactory bulb. Semin Cell Biol 5-1:65-74.
  4. Dyson, G.M. The scientific basis of odour . Chem Ind 57: 647-651.
  5. Wright, R.H. (1982) The sense of smell CRCpress, Boca Raton, Florida, USA.
  6. Turin, L, and Yoshi, F. (2003) Structure-Odor Relations: a modern perspective in Handbook of Olfaction and Gustation Second Edition. Doty RL, Ed. New York Marcel Dekker.

 

 

 

Fire

Leaf lamina. The leaf architecture probably ar...
Image via Wikipedia

In an issue of New Phytologist, Claire Belcher provides a wonderful summary about work by Bond and Scott (from same issue) on the ecological role of fire in the spread of angiosperms (flowering plants). During the Cretaceous (145.5 to 65.5 mya, ending with extinction of dinosaurs), angiosperms were fast growing, weedy and largely understory herbs, shrubs and small trees more likely to colonize edge or disturbance sites. An improved plant vascular system, including a large increase in leaf vein density, doubled the photosynthetic rate and increased leaf mass in comparison with ferns and gymnosperms, which were the dominant land plants at the time. This adaption allowed angiosperms to work more efficiently fixing carbon from falling CO2 levels of the period. Fire also appeared to play a vital ecological role in the spread and ultimate dominance of flowering plants on earth.

Fossil charcoal demonstrated the presence of fire in the early ecosystem. The combination of  highly flammable detritus from weedy angiosperms and increasing atmospheric oxygen levels created an angiosperm-fire cycle equivalent to modern prairie fire cycles. Once an ecosystem was fire damaged, the faster growing angiosperms out-competed both gymnosperms and ferns. The early fossil records indicate that fire activity was greater during the Cretaceous than in previous epochs. However, when oxygen levels dropped about 56 mya, fire-cycles decreased and angiosperm-dominated forests, such as tropical forests, expanded.

In fact, the resulting layers of charcoal helped preserve the fossil record of dinosaurs’ last days. Researchers were then able to predict what the Cretaceous forests looked like.