(1)(a) A proton gradient can be analyzed using fluorescent dyes whose emission intensity profiles depend upon pH. One such dye, useful for measuring the pH gradient across mitochondrial membranes, is called BCECF. When the lipid soluble form of this dye, BCECF-AM, is incubated with isolated mitochondria, it passes easily into the matrix. There, mitochondrial esterases cleave off the lipid group (AM), yielding BCECF which is membrane-impermeable and consequently trapped in the matrix. The effect of pH on the emission intensity of BCECF is shown in the figure below.
(i) Vesicles containing the electron transport chain were made from isolated inner mitochondrial membranes and treated with BCECF-AM. Excess dye was removed and the vesicles incubated in a physiological buffer containing ADP and oxygen. The fluorescence of the BCECF trapped inside the vesicles gradually decreased in intensity. What does this indicate? Explain why an increase in intensity would be expected and what the observed decrease might indicate about the vesicular preparation.
(ii) When the same vesicles were treated with ATP rather than ADP and dilute HCl was added to the buffer, ATP hydrolysis was observed. Explain why this observation is predicted.
(b) A C3 plant and a C4 plant are placed in a sealed glass enclosure and constantly exposed to light. After a few days, the C3 plant begins to die. Explain, with the use of diagrams, why the C4 plant survives and the C3 plant does not.