Rhizosphere Influence on Plant Medicine

Einjähriger Beifuß (Artemisia annua)
Artemisia annua
Wikipedia

Mycorrhization leads to nutrient and information flow, often in both directions. The plant root supplies sugars to the fungus, while the fungus induces Jasmonic Acid biosynthetic enzymes in the plant, leading to an increase in jasmonate ­ levels that enhance the accumulation of soluble sugars in plant root and the production  of plant root defense compounds.

From a research article,  the presence of mycorrhizal fungus, Glomus mosseae and nitrogen fixing Bacillus subtilis on the roots influenced the levels of plant biomass growth, and the yield of an important medicinally active phytochemical, artemisinin, from Artemisia annua L and used as an anti-malarial treatment.

Gabriele et al. (2016) investigated the effect of mycorrhizal soil inoculation of various Sangiovese wine grapes and found the presence of the fungus increased levels of 14 polyphenols compared to un-inoculated plants. Here the presence of symbiotic relations in the soil altered the phytochemical makeup of fruit.

So how are the plant roots attracting mycorrhizal symbionts? Plant produced flavanoid compounds accumulate at root tips/cap and make up a large portion of root exudate (the portion of the root sap excreted to the external environment). These phytochemicals are easily modified and their biosynthesis is triggered  by transcription factors, which suggests a role as elicited signal compounds – compounds that are made specifically in response to conversation from rhizosphere fungi and bacteria. Interestingly, their presence in the rhizosphere soil triggers mycorrhizal fungi to explore their surroundings (Hassan and Mathesius, 2012), perhaps increasing the likely hood of contact with plant roots.

Given the high price of American wild grown ginseng, the ecological influence on ginsenoside formation, and ultimately, the therapeutic value, points to optimizing the rhizosphere cross talk by way of forest farming.

The highest ginsenoside content occurs (from highest to lowest) in the root hairs > lateral roots > cortex > interior taproot (Li and Wardle, 2002), exactly where we should expect a chemical conversation to occur.

Within this class of compounds we designate as ginsenosides, two molecular forms are dominant, protopanaxadiols and protopanaxatriols. Data from two different papers (Zhu et al., 2004: Wang et al., 2010) compared levels of diols and triols in different species and sources of ginseng. American ginseng (Panax quinquefolia) had higher levels of the triols (especially Rg1) compare to Chinese ginseng (P. ginseng), which had higher levels of diols (especially Rb1  Rd).

Structures-of-ginsenosides-from-Panax-ginseng-Glc-glucose-Rha-rhamnose-Araf
Li, H, Lee, JH, and Ha, JM. (2008) Effective Purification of Ginsenosides from Cultured Wild Ginseng Roots, Red Ginseng, and White Ginseng with Macroporous Resins. Journal of Microbiology and Biotechnology. 18(11):1789-91. DOI: 10.4014/jmb.0800.192

Comparing wild grown versus cultivated plants within each species, a similar pattern emerged, with wild plants showing a higher concentration of triols (especially Rg1  Re), while cultivated plants had higher concentration of diols (especially RbRb2).

James, et al. (2013) investigated levels of diols and triols in wild sourced P. quinquefolia leaf and root  in a North Carolina collection, finding that there was no relationship between age and ginsenoside content. However total ginsensosides were higher in the leaf, as was Rb2 and Rd (diols), In the root tissue, Rb1(diol) and Rg1 (triol) was found to be higher.

This has implications for how we “farm” medicine and speaks to a long held tenet; complex interactions in native ecologies, including the soil,  produce medicinal plant crops that are more biologically active. Farm versus wild grown ginseng is only one example. What’s been your experience as a imbiber, herbalist, researcher, plant grower or manufacturer?

 

Do Plant Roots Talk to Leaves?

Arabidopsis thaliana
Arabidopsis thaliana (Wikipedia)

Surrounded by material excreted (exudate) by their own root border cells, the growing root tips (apical region) of plants move through soil regions where important biological interactions occur with a community of soil microbes. This exudate not only helps define the soil microbiome (microbial community), but also changes the physical and chemical characteristics of rhizosphere soil.

Root-tip-tag
Root tip (100×) 1. Meristem 2. Columellae 3. Lateral part of the tip 4. Dead cells 5. Elongation zone (Photo: SuperManu – Clematis)

Hiltpold et al (2011) provided evidence of  systemic, volatile signals in maize roots in response to herbivore attack. From 2013 research on Arabidopsis suggests that soil microbes can alter plant leaf chemistry to inhibit insect feeding. They posited a role for microbial-derived volatile organic compounds acting as a deterrence signal, and noted the presence of Actinobacteria, Firmicutes and Proteobacteria in soil and within Arabidopsis root tissue.In a 2013 Tansley Review, Turnbull and Lopez-Cobello noted that despite localized cellular communication found in the root apical meristem, communication via vascular transport to the rest of the plant did not seem to occur. That left me wondering how plant roots communicated changes throughout the entire plant (systemic).

Those microbes are often associated with “soil odors”. On a sensorial level,  “smelling” the earth may help us appreciate the complex, unseen communication happening under foot.

Are All Plants Carnivores?

Fungal species of the Metarhizium genus colonize most land plants and help provide nitrogen to the plant root. The nitrogen source is unique – insects that the fungus has pathogenized and killed using enzymatic degradation of the insect’s shell.

200px-Metarhizium_anisopliae_infected_cockroach_(PLoS)
Insect infected with Metarhizium spp.

Mike Bidochka of Brock University investigated the phenomena by injecting labelled nitrogen into Galleria mellonella larvae (moth). They buried the larvae in soil and separated the larvae from either beans (Phaseolus vulgaris) or switchgrass (Panicum virgatum) plants using a screen with pores large enough for fungal mycelium to grow through but small enough to prevent plant root growth.

Fourteen days later, they found labelled nitrogen made up more than a quarter of nitrogen found in plant root tissue. Insects Larvae with labelled nitrogen not infected by the fungus did not act as nitrogen sources for the plant.

Good evidence for an ecosystem rich in biota, rather than one where selective human inputs alters it into a simpler set of relationships. In most cases, the soil environment becomes less sustainable.

Plant Chemical Out Posts

Flower of Garden Strawberry (Fragaria ×ananassa)
Image via Wikipedia

The search for chemical mediators in plant root rhizosphere interactions with symbiotic and pathogenic organisms found in the soil continues to generate interesting research. Martha Hawes group at the University of Arizona reported on the role of sugars, proteins and small molecules found in root cap secretions – a mucilaginous mixture that covers the growing root tip and “converses” with the surrounding matrix of living organisms. The cap is rich in root border cells, which detach from the growing root tip. Curlango-Rivera et al (2010) provides us a bit more detail about which metabolites are biologically active. Neither sugars nor amino acids triggered root growth or border cell production. Transient exposure to biologically active concentration levels of the isoflavonoid pisatin, a phytoalexin, stimulated root border cell production but not root tip growth. I wonder if inhibition of root elongation may “reset” plant growth patterns as root border cells, acting as chemical sense organs, define the nature of the environment?

A second paper used histochemcial methods to profile root metabolites in plants from the Rose family (Hoffman et al., 2010). They found flavan-3-ol molecules in the root tip and border cells. Their findings suggest that the distribution of flavan-3-ols in Fragaria and Malus is under tight developmental control. These molecules are found in plants as catechin and epicatechin derivatives and in long chain (polymeric) form. They influence the taste and medicinal potential of green tea and wine, to name a few well-known plants. Previous researchers summarized their role in chelating toxic cations (metals) in the soil, establishing mycorrhizal interactions and priming plant root defense. This paper suggests a role in the transport of the long distance plant hormone auxin, which would link the chemical cross talk at root border cells with responses that occur in tissue distal to root tips. Hoffman’s research lacked a clear distinction of whether the monomeric or polymeric flavan-3-ol forms where the active species. This has plagued plant research for some time, since the analytical methods for detecting the polymeric forms have been crude and ineffective. All of their samples were from a botanical garden. I wonder if the flavan-3-ol profile would differ compared to native wild grown species?

References:

  1. Curlango-Rivera, G. et al. (2010) Plant Soil 332:267-275
  2. Hoffmann, T. et al. (2011) Plant Biology, 13: no. doi: 10.1111/j.1438-8677.2011.00462.x

Ecological Rational for Multiple, Plant Secondary Compounds

(Gossypium L.)
Image via Wikipedia

As I’ve studied medicinal plants, one intriguing question keeps cropping up – what is the biological rationale for plants investing in several classes of structurally varied plant secondary metabolites?. Certainly this question drives a number of researchers in the field of plant chemical ecology. Edwards et al (2008) provided some striking evidence from their study of bacteria resistant cotton (Gossypium hirsutum).

They detected flavonoid pigments, chrysanthemin and isoquercitin, at unusually high levels in epidermal tissue of young leaves in response to Xanthomonas infection. These cells clustered around infected cells that had died because of the plant’s hypersensitive resistance response.

A very different chemical, a  sesquiterpene, 2,7-dihyroxycadalene, acted as a light activated phytoalexin, destroying both bacteria and the infected plant cell. So what were the flavanoids doing to help out? The presence of the flavanoids in surrounding cells appeared to filter sunlight, limiting the generation of free radicals resulting from light activation of 2,7-dihyroxycadaline. How’s that for compartmentalizing your response to friends and enemies?

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