1. Introduction
For decades, discoveries have been reported in the series,
Having established a history of consistent responses to these substrates, we had often taken note of significant differences that were clearly distinguishable to the naked eye; and so we sought methods to photodocument the events by developing systems for cultivation in glass microbeads (μBeads). Thus, we present images of plants treated with indoxyl-β-D-glucoside (IG) as compared to controls. Previously, significant responses of plants to IG had been reported [15,16]; therefore, in our applications of μBeads, the purpose of this section is to exhibit responses of representative plants without further statistical treatment. In addition to serving as solid support media, μBeads refract light to the improvement of photosynthetic efficiency. Not only does the boost to solar intensity from μBeads have the potential to improve productivity, when increased to saturation, it can have the opposite effect of inhibiting growth by photorespiration. Therefore, in consideration of the critical balance that must be achieved, we cultivated plants in μBeads with safeners, selecting appropriately structured substituted sugars.
α-Glycosides have higher binding affinities to lectins over β-glycosides, therefore, we undertook experiments comparing mixed α-and β-anomers to α-mannosides. Mannose polyacetates and methyl-α-D-mannoside were applied to plants because they are closely related to compounds for which we had established dosing. Additionally, responses to low concentrations of arylglucosides, such as IG, provided a starting range of dose requirements for an arylmannoside; and consistent with our hypothesis for specific affinities of lectins, we discovered the highest potencies with μM α-mannosides.
2. Materials and methods
Plants were cultured in research facilities according to previously described methods [1]. Consistency of response to treatments was achieved by supplementation with chelated Ca and Mn. Solutions for foliar applications included phytobland surfactants, but formulas for roots did not. Controls were placed in the same location and all plants were given identical irrigation, fertigation, and handling. Plants were matched to control populations, treated within a week of emergence of cotyledon and true leaves. After treatment, individual plants were scheduled for harvest and analysis. For biomass, plants were dried overnight and weighed. The performance of compounds was evaluated by comparing statistical means of individual dry weights of shoots and roots. All plants were regularly given modified Hoagland water-culture nutrients [17]. Foliar spray applications of identical volumes, either 100 or 186 liters/hectare (L/ha), were mechanically applied. Manual sprays were spray-to-drip volumes of approximately 800 L/ha. For all populations, means of different treatment groups were compared using two-tailed Student’s t-test with
3. Results
The investigations include summaries of previously described experiments with poly-substituted glycopyranoses formulated with nutrients [1]. Manual spray-to-drip foliar treatments were applied to even stands of 5 cm tall radish, as follow: Nutrient Control with 1 g/L surfactants; and 0.3 mM TMG and 1 mM TMG with 1 g/L surfactants. Foliar applications of 1 mM TMG to radish shoots resulted in a significant (n=36; ±SE 0.07;
![](http://cdnintech.com/media/chapter/45009/1512345123/media/image1_w.jpg)
Figure 1.
Foliar applications with low concentrations of polyacetylglucopyranoses, 3 mM MPG and 4 mM TAG, were comparable to treatments with high methylglucosides, 309 mM MeG, resulting in significant shoot enhancements over Nutrient Control. Error bars indicate ±SE.
Treatment of rice with the application of MPG to roots was compared to MeG. Roots exposed to formulations of 500 μM MPG and 50 mM MeG showed significant (n=27;
![](http://cdnintech.com/media/chapter/45009/1512345123/media/image2.png)
Figure 2.
Treatment of radish by 100 μM pentaacetyl-α-D-mannpyranose (MP 100) and 1000 μM tetraacetyl-D-mannopyranose, mixed α/β-anomers (TAM 1000) with nutrients resulted in enhanced whole plant mean dry weight over that of the nutrient Control after 2 days. The α-mannose was more potent than the mixed α/β-anomers. Error bars indicate ±SE.
Owing to enhanced growth and deeper pigmentation in response to treatments with mannosides, we sought a higher potency response, therefore, undertaking rapid radish assays with methyl-α-D-mannoside. Soon after first morning light, exposure of radish sprouts to 500 μM MeM resulted in notable greening of the cotyledon leaves within ~24 h. After 48 h, sprouted germlings treated to 25 μM to 500 μM MeM showed advanced growth responses as compared to Nutrient Control, roots and shoots showing robust enhancement of growth over the nutrient Control, as follow: Application of 500 μM MeM to radish sprouts resulted in statistically significant 11% enhancement of mean dry weight (n=10; 10.3 mg) of whole plants over nutrient Control mean dry weight (n=10; 7.9 mg;
![](http://cdnintech.com/media/chapter/45009/1512345123/media/image3.png)
Figure 3.
Immersion of radish sprouts in methyl-α-D-mannoside (MeM) resulted in significantly increased whole plant mean dry weights of approximately 17% and 12% over the population of the Nutrient Control in 48 h. Error bars indicate ±SE.
![](http://cdnintech.com/media/chapter/45009/1512345123/media/image4.png)
Figure 4.
Treatment of radish with methyl-α-D-mannoside, right, showed enhanced pigmentation and general growth as compared to Nutrient Control, left. By harvest time on the 2nd day, expansion of treated cotyledon leaves and roots was clearly advanced over the Control. Scale bar = 1 cm.
Early on at 24 h, rapid responses were exemplified by visual comparisons of treated and control radish, shown in Figure 4. In one day, treatments with 500 μM MeM, right, showed deeper pigmentation, longer roots and larger expansion of cotyledon leaves as compared to the Nutrient Control, left.
Experience with IG now guided the next experiments to test another arylmannoside,
![](http://cdnintech.com/media/chapter/45009/1512345123/media/image5.png)
Figure 5.
Treatment of radish sprouts in 10 μM
Representative selections from the population treated with an arylmannoside are compared to a nutrient Control, exhibited in Figure 6. A radish germling treated with 10 μM APM, right, showed longer roots and larger expansion of cotyledon leaves as compared to the nutrient Control, left. Also, healthy root hairs are evident. In this experiment, we established the highest potency of the currently tested series of compounds and the growth responses that resulted may be attributable to the specific binding affinities of α-mannosides to lectins.
![](http://cdnintech.com/media/chapter/45009/1512345123/media/image6.png)
Figure 6.
Within 2 days, treatment of radish sprouts by 10 μM
![](http://cdnintech.com/media/chapter/45009/1512345123/media/image7_w.jpg)
Figure 7.
Root hairs of coleus after propagation in 500 μm μBeads are shown. A dip in water rolled μBeads off of roots, leaving the plant intact. The true color image, left, shows root hairs covering the top two-thirds of the white root; and the inverted color image, right, displays the root hairs in silhouette.
Paperwhite narcissus, was cultured in 700 μm μBeads in clear plastic 11 cm tall cylinders with <700 μm diameter perforations for drainage. Roots and shoots are exhibited in Figure 8.
![](http://cdnintech.com/media/chapter/45009/1512345123/media/image8.jpeg)
Figure 8.
Cultivation of paperwhite narcissus in μBeads show a representative nutrient control, left, with a crown of roots up to ~5 cm in length around the basal plate; and, by comparison, when treated with IG, right, with roots elongated ~7 cm.
![](http://cdnintech.com/media/chapter/45009/1512345123/media/image9_w.jpg)
Figure 9.
The index of refraction of µBeads determines the paths of beams of light. A µBead with a high index of refraction, approximately 1.9, sends light back in the general direction of its source, top, a phenomenon known as reflex reflectivity. A µBead with a lower index of refraction, approximately 1.5, may send light out at approximately a right angle to its approach, bottom. In each diagram, the symbol for a point source of light is a triangle in a box, labeled, “Beam of Light;” The circle labeled “Glass Microbead” represents a single µBead; and “Refraction” of a beam of light through the µBead follows the direction of the linear black arrows. Under environments with diffuse lighting, a µBead with a lower index of refraction may be a practical consideration.
![](http://cdnintech.com/media/chapter/45009/1512345123/media/image10.png)
Figure 10.
An aura above a layer of µBeads is shown through polarizing filters. The spectral halo, best described as a three-dimensional rainbow of colors, was the result of upward projections of light by refraction through millions of µBeads spread in a 2 – 3 mm layer over a flat 1 m2 level concrete area. A 30 cm ruler spans the diameter of the circle of light and the black silhouette is of the author’s camera and forearm. The hemisphere is brightest toward its center; moreover, all points of the 1 m2 covered with µBeads were approximately 20% higher in PAR intensity than adjacent surfaces.
4. Discussion
Innovative glassware has been a hallmark of research in photosynthesis and the application of µBeads to refract light to the foliage emphasizes an integral role. At the start, we were faced with several problems; for example, the raw material source of μBeads, recycled sodalime glass, is alkaline. In practice, we found that the smaller the µBead, the larger the relative surface area from which to extract native alkalinity; and their value in daylight had not been considered previously. As pH-stability became an important consideration, it became evident that the largest µBeads would be the preferred media for green plants. Treatment of µBeads with nutribead solution overcame the alkalinity problem while providing a buffered environment for cultivation. Continuous fertigation is a means of stabilizing the medium; and, ideally, automated pH controllers may be implemented to efficiently meter flow rates in a manner that permits high density planting. Such is the case exhibited in Figure 11, showing five paperwhite narcissus plants in a small container with their bulbs nearly apressed. As well, dense cultivation is applicable to protistans as previously demonstrated on “Showa” [1] where frequent flow through of a pH-adjusted nutribead solution is matched by even drainage. Features of daylight enhancement are demonstrated in Figure 10, and because I was enhanced, application of µBeads to crops may entail broadcasting a thin 1-10 mm layer over the ground. As the index of refraction may be specified to direct light at different angles, µBeads of a lower index of refraction may be useful to start crops at subpolar latitudes during seasons for which the angle of solar illumination is low and bending light to a wider angle may distribute illumination advantageously. The application of µBeads in conjunction with glycoside formulations may be requisite to the continued growth of plants exposed to saturated-I, whether or not the overexposure is intentional. It is also important for this system of dual treatments with µBeads and glycoside formulations to maintain a soil at a pH that is amenable for growth. Clearly, for the cultivation of plants, µBeads may be of benefit significant enhancements of ambient light may be achieved by refraction through a multitude of glass spheres.
Applications of polyalkylglucopyranose to shoots of radish resulted in significant root enhancements over controls and, conversely, applications of polyacylglucopyranoses to roots of corn resulted in significant increases of shoots as compared to controls. Similar to findings of our previous experiments with C1 fragments and various glycosides [8,13,15], polyalkyl- and polyacylglycopyranoses required supplementation with nitrogen for significant improvements of growth. The production of ninhydrin-stained products may be from incorporation of nitrogen into protein, drawing attention to lectin as a protein complex from which stores of glucose could be displaced repeatedly by chemical competition with a glycoside. Lectin must be abundant and ubiquitous because, as we have found that Canola and corn respond to treatments of substituted glycosides, lectins occur in C3 and C4 plants. Moreover, not only do plant lectins bind β-glycoside, they bind preferentially to the α-anomer. As much as a quarter of the protein content of seeds and up to ten percent of the protein content of leaves may be attributable to lectins; however, even with such abundance, the provenance of vacuolar lectins was that they served no endogenous role in plants [19,20]. Notably, plant lectins have structural requirements for specific divalent cations to bind sugars [21] and we are currently confirming these requirements with subtractive formulations of corresponding plant nutrient in conjunction with applications of glycosides to plants. The results of our current investigations are consistent with the highly specific binding affinities of mannosides to lectins, the corresponding potencies indicative of their tendencies toward proportionally higher orders of binding to lectins than for glucosides.
![](http://cdnintech.com/media/chapter/45009/1512345123/media/image11.jpeg)
Figure 11.
Five bulbs were planted in close proximity and the paperwhites blossomed while cultivated in 700 μm µBeads. Roots showed through µBeads in the bottom half of the container. Colors from fluorescent illumination contributed to the blue and red hues of the moist µBeads that filled the container.
A case in point, the lectin from
5. Historical note
Steps toward management of the photosynthetic ecosystem were taken when coauthor Benson applied the first available 14C to plants [24, 25] and, most certainly, one of the great joys of life is to have made such extraordinary contributions early in the atomic era. For a time, Benson held the entire concentrated supply of 14CO2 because these were the most rare of all materials. Only the eminently prepared and bravest knew how to handle manmade atomic particles and this required the creation of equipment that had never been known before. For example, when Benson designed the “lollipop” to feed algae 14CO2 with even illumination [1] he developed a method for the “atomic culture” to quickly drop into methanol to stop the reactions at every step of The Path. The keys to the success of this apparatus were attributable to (1) flattening the glass vessel, thus creating an efficient photobioreactor; and (2) enlarging the bore of stopcock, permitting drainage of the entire volume in a second. An original apparatus is exhibited in Figure 13, showing the flat round face and the thin side view resembling a “lollipop” from which the glassware was so appropriately named. For this demonstration, the historically significant flat panel was filled with
![](http://cdnintech.com/media/chapter/45009/1512345123/media/image12.png)
Figure 12.
In The Lectin Cycle, various substrates displace glucose. The
![](http://cdnintech.com/media/chapter/45009/1512345123/media/image13_w.jpg)
Figure 13.
The “lollipop” is a laboratory apparatus for the purpose of cultivating algae to track the path of 14CO2. This first glass photobioreactor was designed by Andrew A. Benson and is exhibited to the left in face view, filled with
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