Back to the Root-The Role of Botany and Plant Physiology in Cannabis Testing, Part II: Plant-Pesticide Interactions and Plant Defense

This article is the second installment of “Back to the Root,” which seeks to explore the effects of plant physiology on topics related to cannabis testing. The term “pesticide” is a broad umbrella that encompasses any substance used to control unwanted or detrimental organisms including plants, insects, and microbes. In an attempt to elucidate the botanical side of plant-pesticide interactions, this article discusses the ways in which chemical pesticides interact with plant physiology, in particular native defense systems. It also draws comparisons between the chemical similarities and differences of synthetic and biologically sourced pesticides (biopesticides). Finally, it explores the unique botanical issues of pesticide interactions in cannabis, a plant with a plethora of novel, poorly understood phytochemicals.

The word pesticide is a broad term that refers to any substance used to control pests, pathogens, or weeds. Pesticides can be defined by the pest that they control (that is, herbicide, fungicide, insecticide, and so on), their chemical structure (organic versus inorganic), their method of action (systemic versus contact), or their source (synthetic versus biological). “Pesticide” can also refer to biological controls such as predatory insects or bacteria that attack a pest organism. For the purposes of this article, which seeks to explore the role that physiology plays in plant-pesticide interactions we will focus on systemic, chemical pesticides, which function by entering plant tissues rather than contact pesticides that interact with the target organism directly. As we will discuss in more detail later on, the fact that systemic pesticides enter plant tissues has important implications for pesticide residue analysis.

In the part I of the “Back to the Root” article series (1), we began with a brief overview of the plant vascular system so that we could discuss the mechanics of plant heavy metal uptake. As a quick reminder, plants have a two-part vascular system that consists of the xylem, which transports water and dissolved minerals from roots to shoots, and the phloem, which transports photosynthates from where they are produced to where they are consumed, commonly referred to as “source to sink” movement. The phloem is also the primary transport system for phloem-mobile nutrients and signaling molecules related to defense responses (2,3). Some important distinctions between these two systems are that the xylem is a one-way system consisting of cells that are dead at maturity, while the phloem has specialized living cells and can transport substances in both directions. We will talk in more detail about the phloem and how it functions shortly, as it relates to the movement of systemic pesticides within plant tissues.

Pesticide Uptake

When considering how pesticides might enter plant tissues, and which system will transport them, it is important to consider the water solubility of the compound in question. Generally speaking, compounds that are more water soluble will translocate (move through plant tissues) via the transpiration stream in the xylem. On the other hand, less water soluble compounds will enter the assimilation stream and translocate in the phloem. Because the transpiration stream requires the tiny pores in the leaf surface, called stomata, to be open, application is best done during the day when plants are photosynthesizing to maximize uptake. In the past, water-soluble systemic pesticides were applied to the soil or root zone in the form of liquids or granules, which makes logical sense because the xylem is a one-way system that moves from roots to shoots. The use of liquid and granular xylem-mobile systemic pesticides used to be more common, however environmental concerns and pest resistance have led to a transition to seed-coating as the primary mode of application (4).

Foliar application of systemic pesticides is usually reserved for compounds that translocate via the phloem. Outdoor cannabis grows might want to be particularly wary of neighbors that are applying systemic pesticides to foliage because there is potential for spray drift and contamination. Phloem-systemic pesticides can also be applied to the root zone because the phloem is an adaptive multidirectional system. This also means that soil contamination of phloem-mobile systemic pesticides can be a potential source of unintentional pesticide uptake. While root uptake of phloem-mobile systemic pesticides is a possible topic of discussion, for the sake of brevity and to avoid repetition of information, this article will focus on systemic pesticides that are applied to foliage.

Phloem Transport

In order for the phloem to transport compounds long distances through the plant body, the system needs to create an energetic force that is sufficient for passive transport, much like the xylem. However, the phloem uses pressure to drive the movement of molecules rather than creating a vacuum effect the way that the xylem does. The most widely accepted explanation for how plants create this pressure in the phloem is the pressure-flow hypothesis (2). Essentially, osmosis is used to create a pressure gradient that causes sugar molecules to flow passively from source to sink. Cells at the source load sugar molecules into the phloem increasing the internal concentration. This causes water to flow passively (via osmosis) into the phloem at the source, increasing the internal pressure. At the other end, sugar molecules are removed from the phloem at the sink, decreasing the internal concentration. This results in a decrease in internal pressure as water flows out of the phloem at the sink. This movement of water into the phloem at the source and out at the sink creates a pressure gradient in the phloem that runs from source to sink. As the phloem sap flows down this pressure gradient it transports the sugar molecules passively from areas of high concentration (source) to areas of low concentration (sink).

It can be confusing to think about the phloem in terms of source and sink, but there is a reason why we can’t use more specific, concrete terms. This general terminology is used because the source and the sink are not static locations; they change depending on the needs of the plant. Sometimes the source might be photosynthesizing leaves, while at other times it might be the roots, say when a plant needs to access carbohydrates that have been stored for a metaphorical rainy day. The same goes for the sink, which might be the roots sometimes or at other times new, actively growing leaves and stems. The very multidirectional nature of the phloem is driven by the fact that the source and sink are relative and can change.

The phloem of flowering plants, including cannabis, is composed of unique cell types that facilitate the movement of photosynthates and signaling molecules. These cells, called sieve-tube elements, are unique because they are alive but have undergone selective breakdown once they reach maturity. What this means is that they are living cells that have lost many of the functional parts, or organelles, commonly found in a cell. If you think of a generic cell, it will contain a variety of organelles and a nucleus suspended in a cellular matrix called the cytoplasm. Sieve-tube elements have lost their nucleus, the “brain” of the cell, along with most other organelles. The remaining organelles are suspended in the cytoplasm, which has been squished to the sides of the cell, forming a layer over the plasma membrane, leaving most of the cell empty so it can act as a transport tube. Specialized structures with large pores called sieve-plates connect the cytoplasm of adjacent sieve-tube elements, which are stacked into systems of sieve tubes, end to end.

So, how does an essentially “mindless” cell control the amount of sugar that enters it to create the driving osmotic gradient, maintain the requisite amount of turgor pressure, and regulate the assimilation stream? Fortunately, sieve-tube elements are not left to manage this complex task alone; specialized cells, called companion cells, are closely associated with sieve-tube elements. In fact, companion cells form from the same mother cell as the sieve-tube element during cellular differentiation. You can think of them as cellular twins that are decidedly not identical. Because they differentiate from the same mother cell they are highly connected to one another via a highway of cytoplasmic threads, called plasmodesmata (2). These threads are located in the companion cell and connect to pores in the sieve-tube element (2). The companion cell uses this connection to act as the control center and “life-support system” of the sieve-tube element (2). The fact that companion cells are very similar in structure to secretory cells is evidence that supports their role as sieve-tube element caretakers.

Thinking back to how the phloem functions to move sugar molecules from source to sink, there is something important I would like to emphasize about the phloem tissue. It is alive. This may seem insignificant but by its very nature, to function the way that it does, the phloem tissues must be alive. In the case of the xylem where the plant is using a vacuum effect to draw water into and up the plant, living cells wouldn’t add any benefit and in fact would be a hinderance to the process. But because the phloem needs to be able to change the source and the sink to meet the plant’s metabolic needs, it needs a selectively permeable membrane that will allow it to actively pump sugar into the sieve-tube elements against a concentration gradient and keep it there. Living cells are, by definition, enclosed by a plasma membrane, which is selectively permeable thus fulfilling a critical criterion that allows the phloem to function.

Pesticide Uptake Challenges

Once a phloem-travelling systemic pesticide has been applied to the leaf surface, it must make its way into the plant and through the leaf tissue to reach the phloem. Leaf surfaces, including the stomatal chamber, are coated in a waxy substance called the cuticle, which helps protect the plant from desiccation. The cuticle consists of an insoluble membrane of polymers, usually cutin a polyester polymer and sometimes cutan a hydrocarbon polymer, studded with and covered by soluble wax platelets (5). Because many pesticides are applied directly to the leaf surface, pesticide-cuticle interactions will determine the ability of a pesticide to penetrate plant tissues. Further complicating matters, the cuticle tends to increase in polarity as you move further into the plant (4). The external cuticle is largely wax-coated, while the interior cuticle has less wax and more pectin, a water-soluble compound you might have experience with if you’ve ever made jelly. Once a compound enters a plant cell, it is dealing with an aqueous and therefor highly polar environment.

Polarity is really best thought of as a spectrum with a variety of related terms that describe other characteristics related to the polarity of a compound. A similar parameter that impacts pesticide permeability is lipophilicity, or how attractive lipids are to a particular compound. A pesticide that is neutral and lipophilic will have better cuticle penetration than either charged or lipophobic (lipid “fearing”) compounds (6). However, if the compound is highly lipophilic, it is more likely to get hung up on the outer layer of the cuticle rather than migrating into the increasingly polar environment of the plant, as would be desirable in a systemic pesticide (5). Ideally, the polarity and lipophilicity of the compound should be carefully balanced to optimize pesticide penetration of plant tissues (5). Compounds that fall in the middle rather than at either extreme will have the best uptake and translocation.

Another issue that foliar-applied pesticides have to contend with is the physical environment of the leaf surface itself. We’ve talked about potential chemical interactions with the cuticle, but pesticides can also interact physically with the leaf surface. The surface of the average leaf is likely to have cuticle protrusions, hairs, and glandular structures. Cannabis has many of these structures, including glandular stalked trichomes, embedded resin glands, and nonglandular trichomes (7). These structures can and do prevent compounds that have high surface tension from making adequate contact with the leaf surface, hampering their ability to enter plant tissues and be systemically distributed (5). Adding a separate compound, such as a surfactant or emulsifier, to pesticides with known high surface tension prior to application can help to mitigate some of these issues by reducing surface tension.

Another complicating factor for systemic pesticides is that the qualities that enhance cuticle permeability, namely moderate lipophilicity and a neutral charge, are different from those that favor phloem mobility. Phloem mobility is enhanced when a compound has intermediate lipophilicity and weak acidity (5). This is thought to be the case because the phloem of many plants is slightly basic, with the pH for a sampling of species ranging between 7.2–8.5 (8). Because little is known about the physiology of cannabis plants, researching the chemical properties of the phloem, particularly pH, could be helpful in predicting pesticidal compounds that are more likely to become phloem-mobile.

Pesticide Research

Researchers of systemic pesticides are already wise to the value an understanding of plant physiology can provide to their work. Systemic pesticides must be carefully designed with plant physiology in mind in order for them to be able to enter plant tissues and operate effectively. Theoretical models have been designed that discuss how to enhance the phloem mobility of a wide range of compounds based on features that render native plant compounds phloem-mobile. In particular, in 1994 Kleier predicted that three types of chemical modifications would increase the phloem-mobility of pesticidal compounds: conjugation with a sugar (sweetening), adding an acidic functional group (souring), and forming quaternary salts from basic parent compounds (salting) (9). How sweetening a pesticide could enhance mobility is fairly intuitive since the phloem is the sugar transport highway of the plant. Adding an acid functional group should enhance phloem mobility because the phloem is a slightly basic environment. The fact that many plant signaling molecules have acid functional groups supports this hypothesis (9). Quaternization will hypothetically lower the pH of a weakly basic compound enough to render it phloem-mobile (9). One caveat of these modifications is that care should be taken to ensure that they do not deactivate the potential pesticide, rendering its mobility a moot point.

One way to potentially circumvent the phloem-mobility hurdle is by producing novel synthetic versions of naturally occurring plant defense compounds. The benefit to this approach is that these compounds should be chemically similar enough to function as their natural counterparts would, allowing them to take advantage of the native defense response infrastructure of the plant. Pyrethroids are an example of a group of synthetic compounds that are chemically similar to the known plant defense compound pyrethrin, which is found in chrysanthemums. While pyrethroids are nonsystemic (contact) pesticides, they are the poster-child for synthetic pesticides based on naturally occurring plant defense compounds, and thus worth mentioning here. Neonicotinoids are another major class of synthetic pesticides based on the chemical structure of the naturally occurring plant defense compound nicotine. Neonicotinoids are an ideal example because they are phloem-mobile, systemic pesticides. The chemical similarity of neonicotinoids to naturally occurring nicotine renders them sufficiently phloem-mobile to be systemically distributed.

Alternatively, instead of using a synthetic molecular analog of a defensive phytochemical, it is possible to use botanically-sourced phytochemicals as biopesticides. However, unlike synthetic pesticides that are usually composed of 1–2 active ingredients, botanically sourced pesticides are often complex mixtures of closely related secondary metabolites that may or may not be active compounds (10). The difficulty of standardizing the active ingredients in botanical biopesticides is another issue that can impact their efficacy (9). Additionally, many botanical extracts and oils that might be used as biopesticides are easily degraded by oxidation or polymerization reactions (10). Botanical biopesticides also tend to be highly volatile as well as sensitive to light, changes in temperature, and air exposure (10). This makes them difficult to store while maintaining their efficacy. Still their use has been increasing significantly over the last decade, making further research to improve them potentially lucrative (10).

As the use of botanical biopesticides continues to grow, more researchers are beginning to see the value in understanding native plant defense systems because they can serve as a source for novel compounds or even just enhance our understanding of how known biopesticides operate. When considering plant defense responses there are two primary categories: induced defenses, which occur in response to a stimuli that indicates a threat, and constitutive defenses, which are always present regardless of attack (3,10). For example, a tobacco plant constantly produces nicotine, making it a constitutive defense compound. Induced responses, that occur in response to a stimulus such as wounding from an insect or herbivore, were originally thought to always be systemic, but more recent research has shown that they can also be localized (3).

Understanding the Plant Vascular System and Pesticides

Having explored how the phloem functions, the chemical and physical barriers that pesticides face in entering plant tissues, and the issues surrounding phloem-mobility of systemic pesticides, there is an elephant in the living room we have yet to touch on. That of the connectivity, or lack thereof, of the plant vascular system. Plant physiologists have long understood that the plant vascular system is not a simple connected highway, but rather a mosaic of connectivity (3,11). Plants are best thought of in terms of integrated physiological units (IPUs) that have a range of connectivity to other units depending on their relative positions (11). Units that are directly connected to one another will have the highest level of exchange through the vascular system, while units that are not directly connected but adjacent will have intermediate exchange, and units that are opposite will have limited exchange (11).

To better understand what is meant by “adjacent” and “opposite” and to help you visualize this concept, imagine that each finger on your right hand is a unit. Let’s say that every other finger is directly connected (your thumb, middle, and pinky all share direct vascular connections). You have the highest level of exchange between these fingers because they are directly connected units. Your thumb is adjacent to your index finger, meaning they are not directly connected but they are close enough to have an intermediate level of exchange. Now look at your left hand and all of its fingers and the same system of direct and adjacent applies to those fingers. However, the fingers on your left hand will have limited connectivity to the fingers on your right hand because they are opposite, lacking either a direct connection or connection by being in the immediate vicinity. This is just an example to help you picture the complexity of plant vascular connections, not an actual representation of any specific connectivity pattern.

The implications of this complexity are staggering. Not only do we have units that interact to varying degrees laterally, but these units also span the plant body vertically, from roots to the shoots (11). This means that the fate of a particular shoot unit will determine the fate of specific roots that are part of the same unit; if the shoot portion of a unit dies back, the roots that are connected will also die back. Further, patterns of vascular connectivity are species dependent and change over the course of a plant’s development (11). Initially, the whole plant is a single unit, but as it ages it will slowly segregate into a mosaic of units with varying connectivity. The take-away is that understanding the vascular connectivity of a plant is critical to predict the movement of compounds.

Another factor to consider is that unlike animals, which will develop to a particular stage and then stop, plants live in a state of perpetual growth. This means that different regions within the same plant are at different developmental stages at any given time. A good rule of thumb that growers likely already know is that the youngest parts of the plant, called meristems by botanists, are located at the ends of branches or roots getting increasingly older as you travel inward towards the main plant body.

To the best of my knowledge, at this time no research has been done to investigate the vascular connectivity patterns of any member of the genus Cannabis. This is a crucial knowledge gap that if remedied could be very helpful in illuminating how compounds translocate in cannabis plants. Further, different chemotypes (that is, high cannabidiol [CBD], high tetrahydrocannabinol [THC], fiber producing, and so on) should be investigated independently, as though they are unique species until the relationships between Cannabis members are better understood. In addition to chemotype, developmental stage is another important variable that is of critical importance to our understanding of how compounds translocate in Cannabis members. If vascular connectivity changes as a plant matures, then applying a pesticide at different growth stages could impact the degree of translocation. This also has unknown implications for translocation of pesticides when cloning cannabis plants. Because clones are sourced from a young portion of the plant, the clone will be in a different developmental stage than the rest of the plant and therefor have a different degree of vascular connectivity.

Pesticide Residue Analysis in Cannabis

In addition to being highly complex in terms of physiological anatomy, cannabis plants are chemically complex as well. They have a plethora of secondary metabolic compounds including flavonoids, terpenes, and of course cannabinoids (6). Earlier we discussed the challenges specific to formulating a phloem-translocated systemic pesticide. How formulators navigate these challenges will also have implications for pesticide residue analysis by analytical testing laboratories. Many of the compounds that are used as systemic pesticides are chemical analogs of defensive phytochemicals, while others are designed to have particular attributes or functional groups that enhance their mobility in the phloem. What this means is that many systemic pesticides will be chemically similar to secondary metabolites naturally occurring in plant tissues. This has serious implications for pesticide residue analysis, especially in a chemically complex plant such as cannabis, that relies on understanding the chemical qualities of a sample to extract, segregate, and quantify analytes. In particular, cannabinoids and terpenes are both hydrophobic molecules, which means that they are extracted by the same solvents used to extract many pesticide residues (12). When a molecule that is chemically similar to an analyte is extracted it can cause interference, making it difficult to quantify the target analyte.

Further, pesticide residue analysis in cannabis requires that samples be homogenized thoroughly enough to rupture cells (12). This is necessary because certain systemic pesticides will be contained within the phloem and thus contained within cells that are bound by a selectively permeable membrane. This means that any other phytochemicals that are present in the sample will be released during homogenization including chlorophyll, another known interfering compound. The implication is that pesticide residue analysis in cannabis is inherently messy for two main reasons:

• because the analytes are chemically similar to many of the compounds that are naturally occurring in cannabis plants; and

• the process of preparing cannabis samples for analysis necessitates the release of interfering compounds.

These interfering compounds have long been a headache for scientists trying to analyze cannabis for pesticide residues. A thorough discussion of the varied issues that can occur during cannabis pesticide residue analysis and the ways that they can be mitigated is better left to analytical chemists, but it is my hope that a better understanding of the physiological aspects of cannabis plants will help these analysts in their research.

Conclusion

In the next installment of this series, we will focus on a specific subset of these interfering compounds, one that the industry has become increasingly interested in: terpenes. The discussion will include a deeper dive into plant defense systems, as many terpenes are defensive compounds. It will also give an overview of synthesis pathways, along with some of the genetic and environmental factors that stimulate terpene production.

References:

1. G. Bode, Cannabis Science and Technology3(2), 26–29, 45 (2020).
2. R.F. Evert and S.E. Eichhorn, Raven Biology of Plants, 8th Edition, (W. H. Freeman, Macmillan, 2013) pp. 546–553, 722–725.
3. R. Karban, Plant Sensing and Communication, (J.N. Thompson, The University of Chicago Press, 2015) pp. 50, 88–89.
4. Z. Vyrzas, Front. Microbiol.7(A 1968), 1–9 (2016).
5. L.A. Norris, USDA Forest Service, “General Technical Report PNW-19,” 1–6 (1974).
6. S. Trapp, Environ. Sci. Pollut. Res.1(A 11), 33–39 (2004).
7. D.W. Pate, J. Int. Hemp Assoc.1(2), A 29, 32–37 (1994).
8. F.C. Hsu and D.A. Klier, J. Exp. Bot. 47, 1265–1271 (1996).
9. D.A. Kleier, Pesti. Sci.42, 1–11 (1994).
10. S. Miresmailli and M.B. Isman, Trends Plant Sci.19(A 1), 29–35 (2013).
11. C.M Orians and C.G. Jones, Oikos94(3), 493–504 (2001).
12. C.B. Craven, N. Wawryk, P. Jiang, Z. Liu, and X. Li, J. Environ. Sci.85, 82–93 (2019).

About the Author

Gwen Bode, B.S., is an aspiring doctoral candidate and botanist with a strong chemistry background. As an undergraduate at Eastern Washington University she investigated the vitamin content of a wild edible plant via HPLC. She has since worked at the front line of the cannabis testing industry, integrating her botanical knowledge with the practical aspects of analytical testing. Direct correspondence to: gwenbode@gmail.com

How to Cite this Article

G. Bode, Cannabis Science and Technology3(4), 36–41 (2020).