Hans W. Heldt

Alkalization of the chloroplast stroma caused by light-dependent proton flux into the thylakoid space

Abstract 1. From the uptake of dimethyloxazolidinedione and of methylamine into the sucrose-impermeable space of intact spinach chloroplasts as measured by siliconlayer filtering centrifugation and from estimation of the size of the thylakoid space by planimetry of electron micrographs the pH in the stroma and in the thylakoid space is evaluated. The reliability of the method is checked. 2. Illumination causes an alkalization in the stroma and an acidification in the thylakoid space, with a ΔpH of about 2.5 between the two spaces, reflecting light-dependent proton transport across the thylakoid membrane. By addition of m-chlorocarbonyl cyanide phenylhydrazone or of nigericin these pH changes are reversed to the corresponding dark values. 3. Beside this there is also some light-dependent proton movement from the stroma across the inner membrane into the external space. 4. The pH in the stroma and in the thylakoid space of illuminated chloroplasts depends on the pH in the medium, whereas the ΔpH across the thylakoid membrane is almost independent from this. 5. From comparison of the pH changes in the stroma and in the thylakoid space it appears that in the thylakoid space there is a buffer with pK 5.5 and in the stroma with pK 6.8 and that the buffer concentration in the thylakoid space is about four times higher than in the stroma. 6. It is discussed that the light-dependent alkalization in the chloroplast stroma may be involved in the regulation of CO2 fixation at the fructose diphosphatase step.

The inner membrane of the chloroplast envelope as the site of specific metabolite transport

The permeability of intact isolated chloroplasts for 3H2O and [14C]sucrose has been measured. Part of the chloroplast 3H2O space is found to be unspecifically permeable to sucrose and other molecules of low molecular weight. The other part of the chloroplast 3H2O space which is impermeable for sucrose is accessible for specific transport of certain metabolites (e.g. 3-phosphoglycerate and malate). n nFrom the comparison of these spaces with the morphological picture as obtained by electron microscopy, the sucrose-permeable space is attributed to the intermembrane space, situated between the inner and the outer membrane of the chloroplast envelope, and the sucrose-impermeable space to the stroma. It is therefore concluded that the outer membrane is unspecifically permeable to metabolites of low molecular weight, the inner membrane being the site of specific metabolite transport.

The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark

1. The pH in the stroma and in the thylakoid space has been measured in a number of chloroplast preparations in the dark and in the light at 20 degrees C. Illumination causes a decrease of the pH in the thylakoid space by 1.5 and an increase of the pH in the stroma by almost 1 pH unit. 2. CO2 fixation is shown to be strongly dependent on the pH in the stroma. The pH optimum was 8.1, with almost zero activity below pH 7.3.Phosphoglycerate reduction, which is a partial reaction of CO2 fixation, shows very little pH dependency. 3. Low concentrations of the uncoupler m-chlorocarbonylcyanide phenylhydrazone (CCCP) inhibit CO2 fixation without affecting phosphoglycerate reduction. This inhibition of CO2 fixation appears to be caused by reversal of light induced alkalisation in the stroma by CCCP. 4. Methylamine has a very different effect compared to CCCP. Increasing concentrations of methylamine inhibit CO2 fixation and phosphoglycerate reduction to the same extent. The light induced alkalisation of the stroma appears not to be significantly inhibited by methylamine, but the protons in the thylakoid space are neutralized. The inhibition of CO2 fixation by higher concentrations of methylamine is explained by an inhibition of photophosphorylation. It appears that methylamine does not abolish proton transport. 5. It is shown that intact chloroplasts are able to fix CO2 in the dark, yielding 3-phosphoglycerate. This requires the addition of dihydroxyacetone phosphate as precursor of ribulosemonophosphate and also to supply ATP, and the addition of oxaloacetate for reoxidation of the NADPH in the stroma. 6. Dark CO2 fixation in the presence of dihydroxyacetone phosphate and oxaloacetate has the same pH dependency as CO2 fixation in the light. This demonstrates that CO2 fixation in the dark is not possible, unless the pH in the medium is artificially raised to pH 8.8.

Specific transport of inorganic phosphate, 3-phosphoglycerate and triosephosphates across the inner membrane of the envelope in spinach chloroplasts.

The uptake of phosphate and phosphorylated compounds into the chloroplast stroma has been studied by silicone layer filtering centrifugation. 1. Inorganic phosphate, 3-phosphoglycerate, dihydroxyacetone phosphate and glyceraldehyde phosphate are transported across the envelope leading to an accumulation in the chloroplast stroma. This uptake proceeds by a counter exchange with phosphate and phosphorylated compounds present there. 2. The transport shows saturation characteristics allowing the determination of Km and V. 3. The phosphorylated compounds transported act as competitive inhibitors of the transport. From measurements of the Km and Ki the specificity of the transport is described. 4. The transport of inorganic phosphate and 3-phosphoglycerate is inhibited by p-chloromercuriphenyl sulfonate, pyridoxal 5-phosphate and trinitrobenzene sulfonate. 5. The activation energy of phosphate transport as determined from the temperature dependence is evaluated to be 16 kcal (0--12 degrees C). 6. It is concluded that inorganic phosphate, 3-phosphoglycerate, dihydroxy-acetone phosphate and glyceraldehyde phosphate are transported by the same carrier, which has been nominated phosphate translocator. 7. Simultaneous measurements of the proton concentration in the medium and the transport into the chloroplasts show that the transfer of 3-phosphoglycerate involves a transfer of a proton into the same direction. 8. Measurements of the pH dependence of the transport indicate that all substances including 3-phosphoglycerate are transported by the phosphate translocator as divalent anions. 9. The physiological function of the phosphate translocator is discussed.

Specific transport of inorganic phosphate, 3‐phosphoglycerate and dihydroxyacetonephosphate, and of dicarboxylates across the inner membrane of spinach chloroplasts

The intact chloroplast is surrounded by two membranes, the outer and the inner membrane. Apart from these are the thylakoid membranes within the chloroplast. Therefore, three compartments of the chloroplast can be visualized: a) the intermembrane space between the outer and the inner membrane; b) the stroma space between the inner membrane and the thylakoid membranes; and c) the thylakoid space located within the thylakoid membrane. From the membranes mentioned the outer was found to be unspecifically permeable to a large variety of solutes, except macromolecules [ 1,2] . Functional studies suggested that 3-phosphoglycerate is able to penetrate the chloroplast [3-61. Direct measurement of the uptake of 3-phosphoglycerate and of malate into the sucrose-impermeable space of chloroplasts has been reported recently [I]. The ability to take up these anions is lost when the inner membrane is disrupted [l] . Thus the uptake of 3-phosphoglycerate and malate is regarded as a transport across the inner membrane into the stroma space. This transport is different from the anion permeability of the thylakoid membrane studied earlier (for references see [7] ).

Light-dependent changes of the Mg2+ concentration in the stroma in relation to the Mg2+ dependency of CO2 fixation in intact chloroplasts

(1) Light-dependent changes of the Mg2+ content of thylakoid membranes were measured at pH 8.0 and compared with earlier measurements at pH 6.6. In a NaCl and KCl medium, the light-dependent decrease in the Mg2+ content of the thylakoid membranes at pH 8.0 is found to be 23 nmol Mg2+ per mg chlorophyll, whereas in a sorbitol medium it is 83 nmol Mg2+ per mg chlorophyll. (2) A light dependent increase in the Mg2+ content of the stroma was detected wjem chloroplasts were subjected to osmotic shock, amounting to 26 nmol/mg chlorophyll. Furthermore, a rapid and reversible light-dependent efflux of Mg2+ has been observed in intact chloroplasts when the divalent cation ionophore A 23 187 was added, indicating a light-dependent transfer of about 60 nmol of Mg2+ per mg chlorophyll from the thylakoid membranes to the stroma. (3) CO2 fixation, but not phosphoglycerate reduction, could be completely inhibited when A 23 187 was added to intact chloroplasts in the absence of external Mg2+. If Mg2+ was then added to the medium, CO2 fixation was restored. Half of the maximal restoration was achieved with about 0.2 mM Mg2+, which is calculated to reflect a Mg2+ concentration in the stroma of 1.2 mM. The further addition of Ca2+ strongly inhibits CO2 fixation. (4) The results suggest that illumination of intact chloroplasts causes an increase in the Mg2+ concentration of 1-3 mM in the stroma. Compared to the total Mg2+ content of chloroplasts, this increase is very low, but it appears to be high enough to have a possible function in the light regulation of CO2 fixation.

Adenine nucleotide translocation in spinach chloroplasts

It was recently reported from our laboratory that intact chloroplasts isolated from acetabularia mediterranea contained endogenous adenine nucleotides, which were exchanged specifically with external adenine nucleotides [ 1,2] . In analogy to the well established adenine nucleotide exchange in mitochondria [3] , this reaction has been called a&nine nucleotide (AdN) tramlocation. For extensive investigations of this exchange reaction, the chloroplasts from acetabularia are not a favourable object, since they have a very low meta-; bolic rate and the algae are difficult to grow. For these reasons, chloroplasts from spinach have been employed in our present investigations to characterize further the AdN translocation of chloroplasts and to evaluate its role in the cell metabolism of the plant.

Accumulation of bicarbonate in intact chloroplasts following a pH gradient

Abstract With silicone layer filtering centrifugation the uptake of radioactively labelled bicarbonate into isolated spinach chloroplasts was followed. This uptake was shown to have the following properties: 1. (a) It is so rapid that the kinetics of uptake usually cannot be resolved. 2. (b) Bicarbonate is accumulated in the stroma. The factor between the internal and external concentrations increases greatly when the pH of the medium is lowered from pH 8.5 to pH 7.0. 3. (c) The accumulation factor is independent of the concentration in the medium for a long concentration range. 4. (d) The accumulation of bicarbonate is increased when the chloroplasts are illuminated. This increase is abolished by the addition of uncoupler. 5. (e) Diamox, an inhibitor of carbonic anhydrase, inhibits the rate of bicarbonate uptake. The activity of carbonic anhydrase was assayed in isolated chloroplasts and in leaf homogenates. In agreement with earlier reports the main activity was found to be located in the chloroplasts. This activity is latent; it can be only assayed if the chloroplasts are osmotically shocked. From these results the following conclusions have been drawn: 1. (a) The inner membrane is impermeable to protons. Light-driven proton transport into the thylakoid space causes an alkalisation of the stroma. 2. (b) The uptake of bicarbonate proceeds via diffusion of CO 2 across the inner membrane. There are no indications for a specific transport of bicarbonate. 3. (c) The CO 2 concentration in the chloroplasts may be equal to the CO 2 concentration in the external space. The distribution of bicarbonate between the two compartments is inversely proportional to the distribution of protons. A possible involvement of carbonic anhydrase and the bicarbonate pool in the stroma in increasing the CO 2 affinity of CO 2 fixation is discussed.

The distribution of metabolites between spinach chloroplasts and medium during photosynthesis in vitro.

1. The formation of metabolites in the stroma compartment of isolated chloroplasts during carbon fixation, and their export to the medium, have been investigated using improved techniques. 2. Rapid separation of photosynthesising chloroplasts from the medium, accompanied by simultaneous quenching of metabolism was achieved by using silicone oil layer filtering centrifugation under illumination. Metabolites were separated by microscale ion-exchange chromatography. Quantitative determination of each metabolite was based on labelling with 32P. 3. It was found that fixed carbon was exported from the chloroplasts only as triose phosphate and phosphoglycerate, and to a minor extent, as pentose monophosphate. The main compounds accumulating in the stroma were hexose and heptose monophosphates and phosphoglycerate. A marked decrease in the concentration of inorganic phosphate in the stroma during the first 5 min of illumination was accompanied by a complementary increase in organic phosphate so that the total amount of phosphate within the chloroplasts remained constant. 4. The concentration difference for phosphoglycerate between the stroma and the medium was much higher than for triose phosphate or inorganic phosphate, although all three compounds are transported across the inner membrane of the chloroplast envelope by the same carrier. It was concluded that the efflux of phosphoglycerate was restricted.

Phosphate requirement for the light activation of ribulose‐ 1,5‐biphosphate carboxylase in intact spinach chloroplasts

Although the fxation of CO* during photosynthesis is a dark reaction, it is generally accepted that some of the enzymes of the reductive COZ fixation cycle are indirectly regulated by light. Fructose and sedoheptulose bisphosphatase and ribulose bisphosphate carboxylase have been identified as major regulatory sites in the reductive CO2 fixation cycle [l-3]. Ribulosebisphosphate carboxylase (EC 4.1 .1.39) converted from an inactive into an active form by reaction with COZ and Mg2+, this activation being enhanced by a pH-shift from pH 7.0-8.5 [4-61. The activation is a relatively slow process with a half-time in the range of l-3 min. The activated enzyme is stable enough to be assayed for 90 s without change of activity [4] . A number of effecters such as 3-phosphoglycerate j 6-phosphogluconate, fructose-l ,6bisphosphate and NADPH have been found to increase the activation of the enzyme especially at pH values below 8.0 [7-91. Using a rapid procedure for the lysis of chloroplasts and assay of the carboxylase activity, the factors