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The Middle Way

James Douglas

M.Sc. Agroecology Candidate, Norwegian University of Life Sciences. Course: Plant Plasticity and Adaptation, PPH-30806, Wageningen Research University.

Introduction  Current agricultural research systems have been

criticized as narrowly focused and discouraging of innovation (Vanloqueren & Baret, 2009). One way of addressing this criticism is to allow a scientific agenda with more input from growers (Warner, 2008). This essay shows how informally generated knowledge, both from accidents during formal research and from farmer observations, can be a resource for advancing scientific understanding.

Accepting these precepts in this essay is an attempt to work within the emerging theoretical framework of Latour’s “circulatory system of science” (Warner, 2008).

Observations are collected from the cannabis industry involving meristem and hormonal manipulation of both fiber and drug-type plants. For fiber-type, there has been an observation made concerning branching as a way to increase total biomass production. For drug-type, industry practices used to increase branching and pharmaceutical yield are interpreted as valid observations. Partial meristem removal, twisting to damage the stem, and bending of the stem are used as examples of techniques.

Auxin canalization theory is used to offer a theoretical interpretation. Further work is suggested, using promising in-vivo monitoring techniques. Finally, manipulation of the meristem with by changing the auxin response via alteration of incoming red/far red ratio is suggested as a practical avenue to explore. This practical area, supported by experimental confirmation of the theory, may aid development of a tool for growers to increase production efficiency by taking advantage of branching behavior.

Basic  Auxin  Theory  One of the classic experiments regarding

branching behavior has been to decapitate a plant and observe behavior. Typically there is activation of dormant meristems. As many gardeners can

describe, this behavior can be used to create a plant with more branches. Currently accepted theory holds that the apical meristem produces auxin which is transported through the stem towards the root. This auxin export is necessary for the meristem to continue to grow and stem concentrations of auxin can prevent export from other meristems, preventing dormant meristems from developing. When the dominant meristem is removed, auxin concentration in the stem decreases and other buds can begin to export auxin into the stem. This acts as a signal and allows them to begin growth. Canalization theory adds that when these new buds begin to grow, auxin transport from the bud happens via a process with positive feedback which results in forming export channels. These channels, once established, quickly conduct auxin into the stem and maintain a stem auxin concentration which prevents other buds from developing.

This mechanism fits observations well, and has been implemented quite successfully via modelling (Prusinkiewicz et al., 2009). Gardeners and mathematical modelers of this mechanism accept that after decapitation new leaders are formed, and once they are formed other dormant meristem development is suppressed. The new leaders establish a sort of dominance, replacing the apical meristem and reducing further branching.

Other theories about hormonal regulation for branching exist, but this paper will focus on auxin canalization theory. Existing theories are not mutually exclusive, and likely interact and coexist.

Observation  1:  Partial  Stem  Removal  In contrast to complete decapitation, partial

removal of the top section of a plant has been less commonly examine. However, “partial decapitation” is used as a technique in the drug-type cannabis industry.

The practice of “floribunding” of “fimming” involves removal of the top 80% of the apical section and is considered useful in drug-type cannabis production.

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Growers believe that, compared to complete decapitation of the apical section, floribunding can lead to increased branching and ultimately higher yields of desired chemical products (Rosenthal, 2010)(Cervantes, 2006).

This technique and observation should be acknowledged because it is commonly observed as effective. However, it should be noted that this practice may technically count as the traditional experiment of complete decapitation because the apical section referred to is actually composed of multiple meristems enclosed by upward-pointing small leaves. When the top of this section is removed, the leading meristem may be completely removed, or simply damaged. Because the meristem is so small, and difficult to see because it is obscured by leaves, what precisely is occurring may vary: it could be that the apical meristem is damaged, or that it is completely removed.

As with the other observations cited here, rigorous science demands that these observations be tested, and described more fully in a more formal research project to draw final conclusions. However, let us suppose that total auxin export may be reduced from the section and try to apply current theory to that scenario.

Observation  2:  Twisting  Twisting the stem, in order to damage it and cause

branching, is sometimes practiced in drug-type cannabis production. The stem is twisted enough to cause damage, without entirely destroying it (Rosenthal, 2010). This kind of twisting damage can cause reduced polar transport from damage to the xylem. When repaired, the xylem is expected to have reduced transport capacity (Fisher & Ewers, 1989).

Observation  3:  Bending  The practice of bending the apical meristem

downwards, without causing any damage to the stem, is also used to increase branching of drug-type cannabis plants. Some authors assert that shoot bending does not induce significant changes in auxin response, but is a gravitropic response involving different molecules (Shimizu-Sato, Tanaka, & Mori, 2009). However, before beginning to draw new mechanisms it should be noted that the PIN3 gene, which has a role in auxin efflux, is expressed in tissues responsible for gravitropism in Arabidopsis (Friml, Wiśniewska, Benková, Mendgen, & Palme, 2002). This is a connection which deserves attention but for which the system connections will not be interpreted here.

A cannabis horticultural manual written for growers states that, “When (the top bud) is positioned at a height below the side buds, it stops producing auxin.” (Rosenthal, 2010). If this is true, it raises the question of how the plant is able to detect being positioned below other buds. Some plants are able to detect the red:far red ratio of light scattered horizontally with considerable sensitivity (Evers, Vos, Andrieu, & Struik, 2006). The phytochrome regulation system is connected to auxin levels: the R:FR ratio of incoming light is known to affect auxin levels in some plants (Almansa, Espín, Chica, & Lao, 2011). Perhaps bending causes reduced auxin levels in the stem, either from a gravitropic or phototropic response.

Observation  4:  Stem  Damage  from  Insects  “Partial meristem removal” has been observed in

fiber-type cannabis. It has been observed that insect attack of the top part of a stem can increase branching and biomass yields of a stand. This observation was made while assessing varieties for their fiber and seed content. Essentially by accident, corn borers (Ostrinia nubilalis) attacked the top of some plants. A corn borer enters a stem by boring a hole in the side and then living for a portion of its life inside the stem. In some cases, the hemp stem was large enough to accommodate the insect without killing the top of the plant (Small, Marcus, Butler, & McElroy, 2007).

It is possible that the corn borer caused damage to the stem and reduced polar transport, including auxin transport. This scenario of insect attack could represent lowered levels of auxin in the stem while maintaining some contribution from the apical section. In this case, apical section damage was not intentional and was arguably beneficial. Although it did result in higher biomass production, branching is usually considered undesirable for fiber production because long fibers are often desired.

Theoretical  Interpretation  The above scenarios may have resulted in

situations which reduced but did not completely eliminate contributions of the apical section to auxin concentration in the stem. They, arguably, resulted in higher yields of desired products.

What could be occurring in these examples is the occurrence of multiple simultaneous canalization events which gives a plant that is “bushy”, with many competing branches, rather than one or two strong leading meristems. Partial decapitation may result in a larger number of less dominant leaders, compared

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to complete decapitation because of canalization kinetics.

Whether multiple canalization events can occur before dominance is achieved by one or more meristems depends on kinetics of canalization and polar transit. Canalization largely depends on initial diffusion characteristics, and polar transit as well as source (apical meristem) contributions impact that. Gradient magnitude is an important part of the canalization scenario, and moderate auxin levels in the stem represent a scenario which might encourage multiple canalization events.

Initial diffusion is the key trigger for a positive feedback loop which comprises canalization events. It also takes time for the decrease in auxin contribution from the stem to affect lower parts of the stem because auxin is not instantly transported downwards. When the apical section is completely removed, auxin levels in the stem are expected to drop dramatically (Leyser, 2011). It may be that meristems immediately below the apical meristem can then form canals and begin to initiate dominance before auxin concentrations fall for lower meristems.

“Partial removal”, in the form of insect damage, twisting damage etc., can be expected to create a less dramatic fall in auxin, which may slow the event to an extent that more meristems establish before any dominant effect is asserted. Figure 1 shows a diagram to help illustrate.

This scenario is supported by modeling (Prusinkiewicz et al., 2009). Figure 2 is taken from the Supporting Information offered by Prusinkiewicz et al, and reports, from a mathematical model.

Before a canal begins to form, diffusion allows auxin to exit the meristem, (Φ, the flux out of the meristem). Diffusion is proportional to the difference in concentration. In the case of reduced auxin contribution from the apical meristem, as the concentration of auxin in the stem (Cj) is lowered a dormant meristem begins to export auxin (initial state may be said to be marked by the hollow circle). At the inflection point (red dot), diffusion is triggering the feedback effect and auxin export rapidly increases.

This can happen simultaneously for multiple dormant meristems. The “experience” of a dormant meristem after apical removal or damage can be visualized as a dot travelling along the curve of figure 2. At the point where the green line turns to red, near (1), cj begins to rise again and the meristem is close to becoming active and asserting dominance. This is a critical point. In the case of fast auxin transport down the stem, other meristems experience the same environment and respond in the same way arriving at a similar situation together. For two or more competing meristems, if both of them cross this critical point before cj=10 they are on the path together for co-dominance. All of the meristems in this situation will likely canalize and begin to grow before dominance is asserted.

Figure   1:   Proposed   auxin   concentrations   for  complete     (left)  and  partial  decapitation  (right)  with  auxin   concentration   profile   in   stem   at   t   and  subsequent   scenario   at   t’   as   the   pulse   travels   down  the   stem.     Curves   are   simplified,   without   showing  contributions   from  other  meristems,  nor  effect   from  varied  polar  transport  speed.    

Figure 2: Graph generated from the model created by Prusinkiewicz et al. (Prusinkiewicz et al., 2009). Φ represents the auxin flux out of a meristem. cj represents the concentration of auxin in the main plant stem.

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In the event of decapitation, if auxin transit down the stem is very slow, compared to this canalization event, then branches at the top of the plant will experience a low cj before others lower down the stem and dominate before others have the opportunity. If auxin transit down the stem is very fast compared to canalization, then all meristems experience the same environment and will activate together or initiate in sequence depending on factors which are not strictly related to auxin concentrations. This can be visualized by multiple dots travelling along the curve in Figure 2, but they begin at different times and their speed of travel is dependent on their auxin flux Φ.

In reality, the change in auxin concentration resulting from an altered meristem contribution (the auxin pulse) does not travel either instantly or extremely slowly. In this model, the same process of auxin transport is responsible for both transporting auxin down the stem and out of the meristem. The feedback effect which means that material is transported faster at higher concentration. This means not only that a pulse will elongate as it travels down the stem, but also that a gentle change in concentration will travel more quickly down the stem. Therefore, a small reduction in apical meristem concentration creates a more uniform environment between the meristems, allowing them more opportunity to co-dominate.

Diffusion as an initial trigger is also supported by this model, and the equations used by Prusinkiewicz recognize that initial diffusion rates are affected by the concentration gradient. Therefore, a small decrease in stem concentration slows the canalization trigger and allows more opportunity for multiple events to occur before the uppermost meristem asserts dominance.

Lateral auxin transport in the stem has long been recognized, and a mechanism more recently confirmed (Friml et al., 2002). This transport may have a critical role in branching behavior. The model referenced above does not include detailed lateral transport. It can be interpreted as simulating the auxin concentration at the edge of the stem, and not the center since this is the relevant area for the initial concentration to affect the diffusion rate. Any regulation which changes the core/edge distribution of auxin will likely change auxin-dependant branching behavior.

Potential  Outcomes  The kinetics of canal formation may require further

work because canalization theory is relatively recent compared to the basic description of polar auxin transport (Prusinkiewicz et al., 2009). Modern instrumentation based on microprobes using fiber optic cables offer a promising method for in vivo auxin analysis (Podrazky et al., 2007). Perhaps these tools could help replace the method of cutting the stem into pieces for analysis.  

If the practice of reducing auxin export from the meristem is found to be helpful to production, the question of practicality arises. Cutting off 80% of the apical meristem, as recommended for “floribunding” may represent a difficult and laborious task for a large production facility. Exploring the photoreceptor relationship with auxin production may yield a more practical option for reducing apical dominance. The R:FR ratio of incident light onto the meristem, if decreased, could reduce auxin concentration (Wit, Ljung, & Fankhauser, 2015). If an appropriate material can be found and the magnitudes of the phenomenon alight, perhaps a spray paint could be developed which alters (increase, by blocking FR) the R:FR ratio of the apical meristem to decrease auxin production. This simulated “meristem damage” may offer greater control over meristem auxin production, and greater convenience than mechanical damage.

Further  Understanding  Whether or not this analysis ultimately provides a

practical outcome for growers, it should offer inspiration and encouragement to continue exploring hormonal regulation. Hormone regulation and signaling control must ultimately obey the rules of photosynthetic capacity and energy budgets, but offers a promising mechanism to direct processes which do not necessarily come into much conflict with energy budgets. Trading plant height and stem weight for pharmaceutical production is a good example since it offers such an excellent payoff in terms of dollars vs joules. If we can understand these systems then we may be able to direct plants to acclimatize to a situation outside their normal evolutionary context. We may essentially communicated with plants and work with them, adapting our systems to them and theirs to ours in order to meet production goals.

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