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Keywords:

  • hydrophilic agrochemicals;
  • hypocotyl;
  • penetration;
  • permselective;
  • pores;
  • radius

Abstract

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS AND DISCUSSION
  6. REFERENCES

Background

Aerial plant surfaces are covered by a lipophilic cuticular membrane (CM) that restricts the transport of water and small solutes. Non-aerial tissues do not exhibit such a barrier. Recent data have shown that large relative to CM hydrophilic agrochemicals were able to pass at high rates through the non-aerial coleoptile.

Results

A moderately large hydrophilic solute like PEG 1000 with a mean molar volume of 782 cm3 mol−1 was rejected by the non-aerial hypocotyl. Uptake of smaller solutes like urea (46.5 cm3 mol−1) was fast and with 99% after 1 day. Cut-off size estimations suggest a pore size diameter below 1.5 nm.

Conclusion

Aerial and non-aerial CM differ largely in their absolute barrier properties. This difference is related to the absence of embedded cuticular waxes in the non-aerial hypocotyl membrane, which make the CM physically dense and cause low solubility of hydrophilic solutes. The free volume for diffusion at the interface of the non-aerial hypocotyl cuticle to the environment is much larger resulting in higher penetration rates. It is suggested that diffusion through the non-aerial hypocotyl does not proceed in a real channel system with continuous aqueous phase but is more like transport through a filter with restricted diffusion in the pore openings. © 2013 Society of Chemical Industry

1 INTRODUCTION

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS AND DISCUSSION
  6. REFERENCES

Aerial primary organs of land plants are covered by a thin lipophilic film, the cuticle. This cuticular membrane (CM) restricts primarily the loss of water by plants. Many mechanistic studies, evaluating the biology, transport and barrier properties of cuticles have been published during the last four decades.[1-4] The impact of agrochemical physicochemical properties on the often low diffusion through the CM and the measures for overcoming this dense lipophilic barrier are quite well understood.[5, 6]

In contrast, the absence of a cuticular barrier in primary roots and root hairs of maize, barley and pea was confirmed long ago.[7-9] Penetration of agrochemicals through plant roots proceeds with an initially rapid penetration rate, which lasts only for a short period and reflects rapid sorption. This penetration rate is followed by another one that is slower but lasts much longer.[10, 11]

Due to its lipophilic nature, the aerial plant cuticle restricts both penetration of water and hydrophilic actives. Therefore, the control of pests often depends on the transport of systemic agrochemicals and the enhancement by adjuvants.[12-14] In contrast, the penetration of various agrochemicals even in the absence of adjuvants was not limited after local application to the non-aerial part of the coleoptile section of grass stems.[15] Significantly lower penetration was measured when agrochemicals were applied to the aerial coleoptile. These findings as well as the similarities in arrangement and number of cortical cells suggested that the non-aerial coleoptile functions like a root, while the aerial part functionally resembles a stem[15] and this was also anatomically concluded for the hypocotyl bean plants.[16] These results indicate that the non-aerial coleoptile may lack the presence of an effective barrier against penetration of hydrophilic solutes.

In spite of the well-known barrier properties of the aerial CM, the existence of two different parallel pathways for the foliar penetration of lipophilic and hydrophilic agrochemicals has been suggested.[14, 17-20] A hydrophilic pathway or channel was originally suggested due to the high weed control efficiency with commercial formulations of hydrophilic herbicides such as glyphosate or paraquat. However, it is important to note that foliar efficacy of such formulations has been optimised through the addition of adjuvants.[21-25]

The lipophilic pathway is certainly the preferred diffusion path when solute mobility is sufficiently high. While lipophilicity correlates positively with penetration and increases with molecular size, mobilities of lipophilic agrochemicals were found to decrease with the size[26-28] and the limiting barrier is primarily defined by the cuticular waxes.[3] The suggested hydrophilic pathway is supposed to be formed by hydration of polar functional groups within the lipophilic cuticular membrane.[14, 17, 19] Average sizes of the aqueous pores have been estimated by different methods measuring transport kinetics and published by a number of authors.[17, 20, 29-31] The estimations for the radius of aqueous pores range from 0.3 to 2.4 nm and the existence of channel-like structures was assumed rather than porous openings. All values are below the dimensions of pores in plant cell walls which are for non-graminaceous species in the range of 3–5 nm.[32] In bio-membranes pore structures involving proteins with a pore size diameter below 2 nm have been reported.[33]

High rates of penetration of hydrophilic agrochemicals appear to be explained by a porous membrane structure. The penetration through the non-aerial hypocotyl could be an efficient site for rapid uptake and translocation of systemic soil applied crop protection products and/or soil fertilisers. The aim of this paper was to explore the transport properties of the non-aerial primary stem membrane by various techniques and to estimate a potential cut-off size. The molecular diffusion of hydrophilic agrochemicals through the non-aerial hypocotyl membrane of mung bean plants and its contribution to the total solute transport will be compared with those of the aerial CM.

2 MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS AND DISCUSSION
  6. REFERENCES

2.1 Effect of molecular size on the penetration of polyethylene glycols into the non-aerial hypocotyl

The non-aerial part of the hypocotyl of mung bean plants (Vigna radiata) was used for the experiments. With the objective to obtain long non-aerial hypocotyls, seeds of mung bean were sown 15 cm deep into the soil. Plants were pot grown on sandy loam and placed in a climate chamber (VB0714; Vötsch Industrietechnik GmbH, Reiskirchen, Germany) exhibiting constant conditions: 26 °C, 70% relative humidity, 12 h of photoperiod and a photosynthetically active radiation of 200 µE m−2 s−1. Only plants grown for up to 10–15 days after germination were used for the experiments. Mung bean plants were removed from the pot and the remaining soil particles still present on the plant surface were washed off with a jet of deionised water. Afterwards, the plants were dried carefully of any excess water by means of a soft tissue. Next, the initial weight of each mung bean plant was measured using an accurate balance (XP205; Mettler Toledo, Giessen, Germany). Pre-weighed mung bean plants were carefully bent and placed in a 10 × 10 cm flat-bottomed chamber (Camag, Muttenz, Switzerland). The inner part of the chamber was completely covered with wet filter paper. After sealing with Parafilm the chambers were able to reach and to maintain a constant high relative humidity (∼90%), which had the objective of reducing the water loss by evapo-transpiration to a minimum. The bottom of the chambers was filled with various osmotic solutions of polyethylene glycol, where only the bent non-aerial part of the hypocotyl was in contact with them. Depending on the size selectivity of the non-aerial hypocotyl, PEG molecules diffuse across the porous membrane. An increase in mass after initial mass loss indicates net movement of water and PEG molecules into the non-aerial hypocotyl. The test solutions used were PEG 200, PEG 400, PEG 600 and PEG 1000. These solutions were dissolved in deionised water at the same osmotic concentration of 0.3 osmolal (osmotically active particles per kg of water). The Parafilm sealing used to cover the chambers was pierced with the aim of restricting the formation of pressures and to allow a free interchange of gases between the interior and exterior of the chamber. Afterwards, the chambers were introduced into a climate chamber exhibiting the following constant conditions: ambient pressure, 25 °C and 90% relative humidity.

The change in mass of the mung bean plants was measured 1, 6, 24, 48 and 72 h during incubation in the test solutions. Trials for the different intervals were conducted separately in time; however, at each interval all the PEG test solutions were tested on the same day. Before measuring the final weight, mung bean non-aerial hypocotyls surfaces were well dried from the excess of the test solution using a soft tissue. For each test solution as well as for each time interval specified above, at least eight replications were carried out. Due to the high osmolality, initially the water diffused out of the mung bean plants. Depending on the size selectivity of the mung bean non-aerial hypocotyl, the different PEG molecules might or might not diffuse across the porous membrane. The change in mass was expressed as a percentage, calculated from the difference between the initial and final weight.

2.2 Penetration of agrochemicals through the non-aerial part of the hypocotyl

After germination on filter paper for about 3 days, seeds of mung bean were sown 2.5 cm deep into the cylindrical experimental vials. These vials exhibited two opposite incisions 3 mm wide and measuring 2.5 cm from the top with Parafilm strapped along the edges. Plants were grown in the same soil substrate and under the same environmental conditions as described above for plants used in the osmotic experiments. Plants between 5 and 10 days after emergence were used for the penetration experiments. Once the plants had been removed from the climate chamber, the Parafilm covering was detached and the soil present in the first 2 cm of the experimental vials was washed off using a jet of tap water. Using a very soft tissue mounted in tweezers, the surface of the now visible non-aerial hypocotyl was dried and cleaned from the remaining soil particles. Radiolabelled hydrophilic agrochemicals dissolved in demineralised water were then applied directly to the non-aerial part of the hypocotyl of the mung bean plants. Agrochemical applications were carried out using a Hamilton #7001 (1 µL) syringe. One droplet (0.5 µL) of the selected radiolabelled molecule at about 160 Bq was applied per plant. Seven [14]C-labelled systemic hydrophilic agrochemicals with different molecular sizes were selected for the experiments (Table 1).

Table 1. Physicochemical properties of selected hydrophilic compounds
NumberCompunds 1 to 6 were labelled with 14C but compound 7 was 3H (tritium)log KOWaWater solubility (g L−1)abMolar volume (cm3 mol−1)c
  1. a

    From BayerCropScience AG.

  2. b

    Water solubility at 20 °C.

  3. c

    Calculated according to McGowan.[36]

  4. d

    log KOW at pH 7.

  5. KOW, octanol-water partition coefficient..

1Urea−2.5959046.5
26-Chloronicotinic acid0.8d5.1372.3
3Glufosinate−4.01500120.6
42-Deoxy-d-glucose−2.69100140.1
5Imidacloprid0.570.610168.3
6Propamocarb hydrochloride−1.31005173.8
73H-adenosine triphosphate0.008450335.5

[14]C-Urea, carbonyldiamide with specific activity 33.63 MBq mmol−1 (Sigma–Aldrich, Seelze, Germany), [3]H-adenosine-5-triphosphate ammonium salt with specific activity 8.46 × 108 MBq mmol−1 (General Electric Healthcare Europe, Freiburg, Germany), 14C-2-deoxy-d-glucose with specific activity 1961.01 MBq mmol−1 (Sigma–Aldrich, 14C-6-chloronocotinic acid with specific activity 1783.52 MBq mmol−1 (Bayer CropScience AG, Frankfurt, Germany), the herbicide glufosinate, 14C-(2R,S)-2-amino-4-[hydroxy(methyl)phosphinoyl]butyric acid (Bayer CropScience) with specific activity 1150.18 MBq mmol−1, the insecticide imidacloprid (Bayer CropScience) 14C-2-imidazolidinimine, 1-[(6-chloro-3-pyridinyl)-methyl]-N-nitro- with specific activity 937.56 MBq mmol−1, and the fungicide 14C-propamocarb hydrochloride with specific activity 1896 MBq mmol−1 (Bayer CropScience) were chosen for the experiments.

In order to check the correct application of the radiolabelled agrochemicals, a water-soluble fluorescent dye (Blankophor; Tanatex Chemicals, Leverkusen, Germany) was added at 0.5 g L−1 to each of the aqueous solutions, which under UV light allows sensitive detection of the exact treated area. As the droplets take roughly 30 min to evaporate, the vials were again covered with Parafilm to negate outside parameters and re-introduced into the climate chamber. The quantity of the radiolabelled molecules that did not penetrate into the non-aerial hypocotyl was determined 24 h after the application using the cellulose acetate technique.34 Cellulose acetate was used at a concentration of 0.1 g L−1 diluted in acetone. The cellulose acetate film strips were then placed in liquid scintillation vials to which acetone (1 mL) and scintillation cocktail (2 mL) (Lumasafe plus; Lumac LSC, Groningen, Netherlands) were added. Radioactivity in the hypocotyl deposit was assayed using a liquid scintillation counter (Tri-carb 1900CA; Packard, Downers Grove, IL,, USA). In order to recover the complete amount of active ingredient that did not penetrate the non-aerial hypocotyl, the cellulose acetate stripping was repeated two to four times on the same treated area. Due to technical reasons the number of repetitions varied from 10 to 15 plants per treatment. The test compounds were applied individually on different days. The total penetration amount was calculated from the amount applied (160 Bq/0.5 µL) minus the residual radioactivity in the strips taken from the hypocotyl. Data were then expressed in percentage of penetration. Their arithmetic means, standard deviations and coefficient of variations were calculated with the statistic program SigmaStat 3.0 (Systat Software Inc., San Jose, CA, USA). To assess mobility and translocation of the hydrophilic agrochemicals applied to the hypocotyl, mung bean plants were placed in the imaging plates. After 72 h of exposure, the imaging plates were scanned, and exposed to the phosphor-imager counter (BAS-1500; Fujifilm Corporation, Tokyo, Japan).

3 RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS AND DISCUSSION
  6. REFERENCES

3.1 Effect of the molecular size on the penetration of polyethylene glycols into the non-aerial hypocotyl

After incubation of mung bean non-aerial hypocotyls in osmotic solutions of different polyethylene glycol (PEG) derivatives, a rapid and strong decrease in mass was observed (Fig. 1). The net movement of water molecules out of the non-aerial hypocotyl was a consequence of the lower water potential of all osmotic test solutions compared to the plant tissue. Whereas PEG solutions exhibits a water potential, ψW, of −0.73 MPa, the non-aerial hypocotyl had a value of ψW = −0.61 MPa. After 1 h the rate of water flowing out of the non-aerial hypocotyl varied only slightly with the molecular size of PEG.

image

Figure 1. Time course of change in mass of mung bean plants incubated in different polyethylene glycol (PEG) test solutions. The bars represent the standard deviation (SD).

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The flow of water was generally rapid, indicating a high water permeability of the non-aerial hypocotyl membrane. However, after 6 h, the rate of mass increase of the non-aerial hypocotyls incubated in PEG 200 was much higher than with PEG 400 and PEG 600. The mass lost by the PEG 200 after 6 h was compensated in non-aerial hypocotyls by about 60% after 3 days. The regain in mass after 3 days was inversely proportional to the size of the diffusing PEG molecule. Accordingly, the water flow back to the tissue parallel to the PEG was fastest with the smallest molecule, PEG 200. With PEG 400 and PEG 600 the net back-flow of water was recorded after 3 days as 28 and 17% respectively in comparison to the lowest value at 2 days. The slope showing the increase of mass with the PEG 200, PEG 400 and PEG 600 solutions between 2 days and 3 days was found to be identical (m = 0.24, where m is is the slope of the line). This indicates that with time mung bean plants incubated in PEG 200, PEG 400 and PEG 600 test solutions will gain weight at the same rate until equilibrium is reached.

The regain in weight by the non-aerial hypocotyls of mung bean plants after a transitional loss with PEG 200, PEG 400 and PEG 600 test solutions suggests that molecular sizes of these osmotica are small enough to diffuse through the non-aerial hypocotyl membrane. In strong contrast, the non-aerial hypocotyls incubated in PEG 1000 did not regain weight even after 6 days and the efflux follows first-order kinetics. Due to the efflux of water from the non-aerial hypocotyl membrane, mung bean plants incubated in PEG 1000 exhibited a decrease in mass by 65% of the initial weight.

The pore size of the non-aerial hypocotyl can be roughly estimated by comparing the sizes of the PEG molecules which were able to diffuse versus those rejected by the non-aerial hypocotyl membrane. Transport through pore-like structures can be characterised by descriptive measures that are worth considering in the light of the current results. The molecular radius (rM) of each of the used PEG samples was calculated using the approach reported by Renkin[35] and when we assume spherical shape the radii of the diffusing molecules can be calculated with Equation (1):

  • display math(1)

where Vx is the molar volume of the diffusing molecule calculated according to McGowan[36] and N is Avogadro's number. Average radii for PEG 200, PEG 400, PEG 600 and PEG 1000 were calculated in the present study to be 0.41 nm, 0.50 nm, 0.58 nm and 0.68 nm, respectively. However, in contrast to the assumption with the Renkin approach, PEG molecules are not spherical in solution but show dynamic irregular conformational changes. The hydrodynamic radius (rH) also assumes them to be sphere-shaped but in addition includes the dynamic structural changes of shape in solution. For the calculation of PEG rH, the Stokes–Einstein equation (Equation (2)) can be used.[37] This equation shows that diffusion (D) depends strongly on the translational movements, which are included in the properties of the rH:[38]

  • display math(2)

where KB is the Boltzmann constant, T is temperature (K), and η is the solvent viscosity (in Kg m− 1 s− 1). If we assume a diffusion step across a sieve with hydrophilic pore openings, then rH as a dimensional parameter would be more relevant than volume based calculations of radius (rM). Values of rH calculated in the study carried by Hämäläinen and co-workers[37] were 0.45 nm for PEG 200, 0.58 nm for PEG 400, 0.68 nm for PEG 600 and about 0.85 nm for PEG 1000.[37] These rH values are higher than those for the rM, (Fig. 2). Comparing the sizes of the PEG molecules, which were able to diffuse against those rejected by the non-aerial hypocotyl membrane, we can infer that the cut-off size for the rapid diffusion of hydrophilic molecules across mung bean non-aerial hypocotyls is around 1.5 nm.

image

Figure 2. Correlation between the molar volumes (Vx) of the osmotic PEG test solution and their radii: (squares), hydrodynamic radius; (circles), molecular radius. Molecular volumes were calculated according to McGowan.[36]

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Due to the close molecular dimensions of both the non-aerial hypocotyl pores and the diffusing PEG molecules, the diffusion process might be affected by solute–wall hydrodynamic interactions and solute sieving. The ratio of solute radius, rH, and pore radius, rP, (Equation (3)) is known as the relative solute size (λ) and indicates the hindrance of diffusion through a porous membrane. For the calculations, the rH mean size of PEG 600 and PEG 1000 was chosen as 0.77 nm for the non-aerial hypocotyl pore radius (Fig. 2).

  • display math(3)

The hydrodynamic interactions depend strongly on the proximity of a solute molecule to the walls of the membrane pore.[39] Due to the large size of PEG 1000 its diffusion was impeded by the non-aerial hypocotyl cuticle. Pores of the non-aerial hypocotyl exhibited a smaller diameter than the diffusing molecules of PEG 1000.

Diffusion of molecules in water-saturated porous membranes depends on intermolecular collisions between water and solute molecules.[40] Therefore, molecular diffusion is not solely related to the pore diameter or to the pore size distribution. Molecular diffusion of an agrochemical will be significant only if pores of the non-aerial hypocotyl are large compared to the mean free path length of the solute molecule. Due to the proximity among solute molecules in solution, they can only move a few angstroms before colliding with each other.[41] In aqueous systems, the mean free path length for most organic substances is about 1 nm.[40] This is the same order of magnitude as the cut-off size found for the non-aerial hypocotyl, making free diffusion in an aqueous liquid of molecules having solute size also in that range very improbable. Since radii of the diffusing PEG molecules lie in the same order of magnitude as the calculated pores of the non-aerial hypocotyl, a so-called ‘restricted diffusion’ exists. This means that even PEG 200 may have exhibited a restricted molecular diffusion rather than free diffusion along an aqueous pathway into the non-aerial hypocotyl. Due to this wall proximity for PEG 200–600, not only drag but increased viscosity of the solvent inside the pore was restricting diffusion versus free diffusion in water. This increase of solvent viscosity is a result of both solvent and solute proximity to the pore walls.[41] For large relative solute size (λ > 0.4), the limited transport can be described by a hindrance factor (H), which is quantified by the empirical Equation (4) developed by Bungay and Brenner.[42] For uncharged molecules such as PEG molecules, these authors were able to combine asymptotic results of hydrodynamic analysis into the algebraic expressions:

  • display math(4)

The asymptotes are: a1 = −73/60, a2 = 77 293/50 400, a3 = −22.5083, a4 = −5.6117, a5 = −0.3363, a6 = −1.216, and a7 = 1.647.

The restricted diffusion through pores of the non-aerial hypocotyl can be seen after plotting the hindrance factor against the relative solute size (λ) (Fig. 3). Only if λ has a value of less than 0.1, the hindrance factor will be at least 0.6 (Fig. 3), which is the threshold for free diffusion in the bulk liquid.[40] Since sizes of the diffusing PEG molecules lie in the same order of magnitude as that of the non-aerial hypocotyl membrane, is much higher than 0.1 and H is much lower than 0.6. This indicates that transport of PEG 200, PEG 400 and PEG 600 molecules through the non-aerial hypocotyl can be characterised as hindered transport.

image

Figure 3. Restricted diffusion of PEG 200, 400 and 600 through pores of the non-aerial hypocotyl. The mean size of the hydrodynamic radius (rH) between PEG 600 and 1000 was chosen as the non-aerial hypocotyl pore radius.

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Based on these results we can conclude that the diffusion of hydrophilic molecules through pores of the non-aerial hypocotyl is size dependent and more size selective than in solution. This information is valuable for evaluating the uptake of water, ions and soil-applied agrochemicals from the soil solution.

3.2 Penetration of agrochemicals through the non-aerial part of the hypocotyl

Penetration of several radiolabelled hydrophilic molecules with molar volumes differing eight-fold (Table 1) was assessed 1 day after the direct application to the non-aerial part of mung bean hypocotyls (Fig. 4A). As in the osmotic studies, the decrease in active ingredient penetration was found to be inversely proportional to the size of the diffusing molecule. The smallest molecule, urea, was able to completely (99%) penetrate the non-aerial hypocotyl after 1 day. But even the large molecule ATP penetrated through the non-aerial hypocotyl membrane at high rates (69% after 1 day).

image

Figure 4. Penetration and translocation of selected radiolabelled hydrophilic molecules. (A) Uptake was measured 1 day after the local application to the non-aerial part of the hypocotyl. Molecular volumes were calculated according to McGowan.[36] Bars represent the standard deviation (SD). Numbers refer to Table 1. (B) Translocation of 14C-imidacloprid 1 day after the application to the non-aerial hypocotyl of mung bean plants. The arrow indicates the area where radiolabelled agrochemicals were applied.

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Although the molecular size of ATP is almost eight times greater than that of urea, ATP penetration was only 0.7-fold slower than urea through the non-aerial hypocotyl. The size dependence is thus quite low and close to Stokes diffusion in liquids. Since no correction can be made for differential solubility in the pore no (relative) diffusion coefficients can be estimated. Also, it is unclear whether the pore wall interaction is higher with urea or ATP since the nature of the wall groups is unknown. Nevertheless, from Fig. 5 it is possible to conclude that even urea, which is not only the smallest molecule used in these experiments but also one of the smallest agrochemicals, followed a restricted diffusion through pores of the mung bean non-aerial hypocotyl. These results suggest a significant pathway of the non-aerial hypocotyl for the uptake of soil applied agrochemicals. Practically all agrochemicals do not have a suitable solute size enabling free diffusion, but exhibit only a restricted diffusion. After penetration through the non-aerial hypocotyl, the radiolabelled agrochemicals were quickly distributed along the different plant tissues. For instance, the autoradiography of imidacloprid after one day showed the fast acropetal translocation, which was found to be typical after application to the hypocotyl (Fig. 4B).

image

Figure 5. Restricted diffusion of the selected hydrophilic compounds listed in Table 1. The mean size of the hydrodynamic radius (rH) between PEG 600 and 1000 was chosen as the non-aerial hypocotyl pore radius (0.77 nm).

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The calculated pore size and the restricted diffusion coefficients for the hydrophilic compounds suggest that the voids are of the same size as for the cuticular penetration of foliar applied products. Penetration studies found that 1 day after foliar application of the hydrophilic insecticide imidacloprid, only 2% of the amount applied was found inside wild oat plants (data not shown) in contrast to the 68% found for the wild oat coleoptile.[15] Glyphosate salts (i.e. trimesium, isopropylammonium, ammonium, sodium) exhibit high water solubility,[43] but similar to imidacloprid, lipid solubility is extremely low.[44] In the absence of adjuvants, the penetration of [14]C-glyphosate (isopropylammonium salt) 2 days after the application on guinea grass (Panicum maximum) and redroot pigweed (Amaranthus retroflexus) adaxial leaves showed an amount of 2.3 and 3.1% of the amount applied respectively.[25] This indicates that even without the presence of adjuvants, imidacloprid as well as glyphosate molecules (although at extremely slow rates), are able to penetrate the aerial cuticular membrane (CM). This suggests that pores of the same size as those found in the non-aerial hypocotyl exist as well in the aerial CM. However, there is no clear evidence for the presence of pores in the aerial CM. The penetrated fraction of glyphosate through the aerial CM is very low and can be explained as a simple diffusion along a driving force with low glyphosate membrane solubility but high outer concentration or solubility in the hydrated deposit, respectively.

The main limiting barrier for the diffusion of water and hydrophilic compounds is formed by embedded cuticular waxes.[3] By treating the plant cuticle with methanol and chloroform, embedded waxes can be extracted.[45] This extraction decreases the viscosity of the cuticle creating what is called a polymer matrix membrane. Permeability coefficients of the matrix membrane were found to be about three orders of magnitude higher than those of the plant cuticle with cuticular waxes.[17] In this case a hydrophilic pathway exists and transport of water and solutes is affected by the presence of polyvalent cations. From our penetration studies we suggest that the non-aerial hypocotyl equals a matrix membrane. The non-aerial hypocotyl did not show barrier properties for the penetration of hydrophilic agrochemicals (Fig. 4A) and probably lacks the presence of the viscous cuticular waxes.

At comparable driving force, penetration of agrochemicals through the non-aerial hypocotyl will depend strongly on the size of the diffusing molecule.[3] The total penetrated amount of radiolabelled agrochemical [1 − (Mt/M0)] was calculated from the amount applied (M0) and the residual radioactivity found after 1 day (t) on the surface of the non-aerial hypocotyl (Mt). The diffusion process corresponds to first-order kinetics and at continuous conditions the slopes of the lines correspond to the constant k*, which can also be considered as an indicator of solute mobility in the hypocotyl. For determining the diffusion coefficients (D) across plant cuticles, the path length of the limiting skin has to be known.[3] However, since the path length of the non-aerial hypocotyl is not exactly known, the constant k* (Equation (5)) is used instead of the apparent diffusion coefficients:[3, 26]

  • display math(5)

The path length is composed by the thickness of the limiting skin (Δxls) and the thickness of the sorption compartment (Δxsoco). In order to compare the size effect on the mobility of agrochemicals across plant membranes, the logarithm of the constant k* is plotted against the molar volume. This dependence can be described by Equation (6):

  • display math(6)

where the y-intercepts (log k*0) represent the mobility of a molecule having zero molar volume and the slopes of the graph (β′) represent the size selectivity of the barrier. Both parameters are used here to characterise the barrier properties of the hypocotyl membrane. With this information the effect of the embedded cuticular waxes in the mature leaf CM can be compared with those in the non-aerial hypocotyl (Fig. 6).

image

Figure 6. Effect of the molecular size (Vx) on solute mobility (k*) through polar cuticular membranes and non-aerial hypocotyls of mung bean plants. Mobility data for lipophilic and hydrophilic compounds across polar cuticular membranes were drawn from Buchholz et al.[28] and Schönherr,[20] respectively.

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The mobility of lipophilic solutes (the diamonds in Fig. 6) was clearly lower than those of the hydrophilic ones across the polar CM (squares in Fig. 6). The size selectivity for lipophilic solutes was much higher than for hydrophilic solutes. The slope and thus size selectivity for the non-aerial hypocotyl membrane with the hydrophilic compounds was even lower and the y-intercept showed the largest value with 0.146 (circles in Fig. 6). Since the y-intercept can be related to the free volume available for diffusion in cuticles and cuticular waxes,[26, 28] we can conclude, first, that the non-aerial hypocotyl lacks a real penetration barrier (the cuticular waxes); and, second, that the free volume available for diffusion is much larger in non-aerial than in aerial membranes. Differences in composition and viscosities of the medium are the reasons for the extremely high penetration rates of hydrophilic agrochemicals for the non-aerial hypocotyl. It may be argued that the low size selectivity due to a more liquid phase is accompanied by a smaller thickness of the transport limiting layer.

The data presented in this study suggest a significant role of the non-aerial hypocotyl for an efficient penetration of soil applied agrochemicals. Clear differences in composition and size selectivity between aerial and non-aerial membranes were shown. The pore size calculations of the non-aerial hypocotyl using osmotica with different molecular sizes gave diameters around 1.5 nm which is in the same size range as those suggested for hydrophilic pathways reported for the aerial CM. The definition of pore size in this context has to be taken with care in either case. Radii of modern agrochemicals are in the range of at least 0.7 nm (e.g. azole fungicides) and thus even higher than carbohydrates (e.g. 0.45 nm for sucrose). This means that such space is generally needed for the permeation through the cuticle including molecules that are moving as solutes solely in the lipophilic domains of the cuticular membrane. This scale of 1.5 nm is lower than for the diffusion in the aqueous cell wall of plants for which higher pore sizes have been reported of about 3–4 nm (range 2–6 nm) in various species.[32] In contrast, in biomembranes, even for pores based on transmembrane proteins, diameters of 2.2 nm in bacterial cells are found,[33] down to only 1 nm for transmembrane leucotoxins in bovine lymphoma cells,[46] or 0.74 nm in human lymphocytes, which greatly increases the transport of hydrophilic solutes. In this context it is interesting that our results with the hypocotyl have shown that PEG 200 (radius ∼0.5 nm) was moving more freely than higher homologues, which was found also for diffusion through channels in biomembranes.[47] Considering these dimensions and the biomembrane thickness of about 5–10 nm, we think that pores in the hypocotyl act more like a sieve and are distinguished by a lower viscosity in the hydrated domains than in the surrounding lipophilic areas.

Only a restricted diffusion appears to occur in the areas accessible for diffusion of hydrophilic solutes. Transport follows a constrained molecular diffusion process rather than free diffusion along an aqueous channel. Similarly, the 40-fold faster penetration of the hydrophilic insecticide imidacloprid through the non-aerial coleoptile membrane indicates that lower diffusion rates across the mature oat CM are not necessarily due to narrower pores, but to the presence of the real penetration barrier with embedded cuticular waxes. The result is, therefore, more a sieve with hydrophilic pore openings rather than a hydrophilic ‘water-filled’ pathway. The cuticular waxes make the aerial cuticular membrane physically much denser, decreasing substantially the free diffusion of hydrophilic compounds. Since the non-aerial hypocotyl is not a permeability barrier like the aerial leaf cuticle and the free volume for diffusion is much higher, the non-aerial part of the hypocotyl can be used as an optimal pathway for the penetration of soil applied low molecular weight agrochemicals.

REFERENCES

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS AND DISCUSSION
  6. REFERENCES
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