Passive Absorption — Ion Trapping and Accumulation

So far in discussing the concepts of passive absorption of herbicides into plant cells, we have been focusing on those properties of the herbicide molecule which play a role in its absorption. Now, we will also consider the properties of the plant cell which have a strong influence on the amount of herbicide that accumulates at a specific cellular location. In this section we will learn those mechanisms by which herbicides can reach high concentrations inside of plant cells.

Typical pH values for the plant cell cytoplasm are about 7.5, while the extracellular and vacuolar spaces are about 5.5 (Figure 7a). This pH gradient or proton motive force (PMF) is maintained by the continual removal of H⁺ to the vacuole or to the apoplast by the proton-ATPases (H⁺-ATPases) located on the plasma membrane and tonoplast, the membrane surrounding the vacuole (Figure 7b)

Once in the alkaline or basic compartments of the cell, the acid (HA) dissociates into its conjugate base (A^-). Because the acid form is no longer present in the cytoplasm, a concentration gradient causes further influx of the acid. This results in the anionic form (A^-) of the herbicide accumulating in the cell. Because the PMF depends on functional proton-ATPases, accumulation of weak acid herbicides requires metabolic energy. It is important to clarify here that although ATP is required to establish the PMF, only passive processes are involved with the transport of the lipophilic herbicide into the cytoplasm; once in the cytoplasm, the acid becomes the anion, creating a concentration gradient for further absorption, and hence, accumulation. This general process of anion accumulation is known as “ion trapping”. By following the link for “ion trapping” in this animation, Herbicide Uptake by Leaves and Cells, you can see the role of herbicide ionization and this process animated (Figures 8a and 8b).

By knowing the physiochemical properties of any given herbicide, we can predict the amount of ion trapping, and thus, predict herbicide concentrations in plant cells. This knowledge is important to consider when designing herbicides if the designer wants them to accumulate near sites of action. The characteristics controlling the amount of ion trapping are the following:

• the membrane permeability of the acidic and anionic form of the herbicide,
• the pH inside and outside of the cell (i.e. PMF, pH gradient), and
• the membrane potential (E).

Mechanistic evidence for passive absorption of lipophilic, ionic herbicides and their accumulation by ion trapping exists only for weak acid herbicides such as bentazon, 2,4-D, clopyralid, chlorsulfuron, imazapyr, and sethoxydim. In contrast to lipophilic, neutral herbicides, weak acid herbicide absorption saturates over time, with internal concentrations becoming greater than external concentrations. This accumulation can be reduced by metabolic inhibitors or anoxia (lack of oxygen). Accumulation is enhanced by an acidic pH external to the cell, suggesting accumulation is energy dependent and is mediated by ion trapping. Higher concentrations of the undissociated herbicide exist at acidic rather than neutral pH (recall the Henderson-Hasselbach equation from the previous topic:  pH  =  pKa  +  log  [A^- ]/[HA] ). The neutral form (undissociated acid, HA) diffuses across the plasma membrane because cell membranes are more permeable to undissociated, neutral molecules (higher K_ow) compared to dissociated (A^-), charged molecules (lower K_ow). These charged molecules are less able to diffuse back out of the cell and thus, accumulate in the alkaline cytoplasm via ’ion trapping’.

Thinking Question:

Which of these will be able to traverse membranes more rapidly, and why?

• R-NH₃⁺,
• R-COOH, NH₂, or
• R-COO^-

The answer is: R-COOH and R-NH2 because both are uncharged with higher Kow values, and therefore are more lipophilic. Because membranes are made up of hydrophobic lipids, they are more permeable to lipophilic molecules. The membrane is also permeable to the charged forms of herbicides, but much less so. Therefore, those herbicides which are hydrophilic or charged, will traverse the membrane at a much slower rate.