Chloroplast biogenesis. Regulation of lipid transport to the thylakoid in chloroplasts isolated from expanding and fully expanded leaves of pea

Abstract

To study the regulation of lipid transport from the chloroplast envelope to the thylakoid, intact chloroplasts, isolated from fully expanded or still-expanding pea (Pisum sativum) leaves, were incubated with radiolabeled lipid precursors and thylakoid membranes subsequently were isolated. Incubation with UDP[(3)H]Gal labeled monogalactosyldiacylglycerol in both envelope membranes and digalactosyldiacylglycerol in the outer chloroplast envelope. Galactolipid synthesis increased with incubation temperature. Transport to the thylakoid was slow below 12 degrees C, and exhibited a temperature dependency closely resembling that for the previously reported appearance and disappearance of vesicles in the stroma (D.J. Morré, G. Selldén, C. Sundqvist, A.S. Sandelius [1991] Plant Physiol 97: 1558-1564). In mature chloroplasts, monogalactosyldiacylglycerol transport to the thylakoid was up to three times higher than digalactosyldiacylglycerol transport, whereas the difference was markedly lower in developing chloroplasts. Incubation of chloroplasts with [(14)C]acyl-coenzyme A labeled phosphatidylcholine (PC) and free fatty acids in the inner envelope membrane and phosphatidylglycerol at the chloroplast surface. PC and phosphatidylglycerol were preferentially transported to the thylakoid. Analysis of lipid composition revealed that the thylakoid contained approximately 20% of the chloroplast PC. Our results demonstrate that lipids synthesized at the chloroplast surface as well as in the inner envelope membrane are transported to the thylakoid and that lipid sorting is involved in the process. Furthermore, the results also indicate that more than one pathway exists for galactolipid transfer from the chloroplast envelope to the thylakoid.

Figure 1

Presence of Tic110 in intact chloroplasts and thylakoid fractions isolated from whole pea seedlings and still-expanding leaves. Intact chloroplasts (C) and thylakoid (T) fractions corresponding to 0.2 (subscript 1), 0.4 (2), and 1 (3) μg of chlorophyll isolated from expanding leaves and whole pea seedlings were separated by SDS-PAGE and immunoblotted with anti Tic110. Thylakoid fractions were isolated from batches of of chloroplasts corrsponding to 100 μg of chlorophyll.

Figure 2

The time dependency of the incorporation of [³H]Gal into galactolipids of pea chloroplasts and their subsequent transfer to the thylakoid. Intact chloroplasts isolated from whole pea seedlings (corresponding to 100 μg of chlorophyll) were incubated with UDP-d-[6-³H]Gal at room temperature for the times indicated and a thylakoid fraction was subsequently isolated. White symbols denote chloroplast fraction and black symbols thylakoid fraction; squares, radiolabel recovered in MGDG; circles, radiolabel recovered in DGDG.

Figure 3

The effects of KF on the transfer of MGDG and DGDG to the thylakoid. Intact chloroplasts (100 μg of chlorophyll) isolated from whole pea seedlings were incubated with UDP-d-[6-³H]Gal for 20 min at room temperature with or without inclusion of 2 mm KF and thylakoid fractions were subsequently isolated. Black bars, Control; hatched bars, with inclusion of 2 mm KF. Mean values and the range from duplicate samples within one representative experiment are presented.

Figure 4

The effects of thermolysin treatment of intact chloroplasts on the synthesis of galactolipids and on their subsequent transfer to the thylakoid. Thermolysin-treated intact chloroplasts (100 μg of chlorophyll), isolated from whole pea seedlings, were incubated with UDP-d-[6-³H]Gal for 20 min at room temperature and thylakoid fractions were subsequently isolated. Cross-hatched bars, Radiolabel recovered in chloroplast MGDG; black bars, radiolabel recovered in thylakoid MGDG; hatched bars, radiolabel recovered in chloroplast DGDG. Mean values and the range from duplicate samples within one representative experiment are presented.

Figure 5

The temperature dependency of galactolipid synthesis and transport to the thylakoid in intact chloroplasts isolated from fully expanded (A) or expanding (B) leaves. Intact chloroplasts (100 μg of chlorophyll) were isolated from the fully expanded two lowermost leaves of 10- or 7-d-old pea and incubated for 20 min with UDP-d-[6-³H]Gal at the temperatures indicated, followed by isolation of a thylakoid fraction. White symbols denote radiolabel in chloroplast fractions and black symbols radiolabel in thylakoid fractions; squares, radiolabel recovered in MGDG; circles, radiolabel recovered in DGDG. Mean values and the range from duplicate samples within one representative experiment are presented.

Figure 6

Positional distribution of radiolabeled acyl groups in phospholipids in intact chloroplasts and in the thylakoid. Intact chloroplasts were incubated with [¹⁴C]acyl-CoAs and a thylakoid fraction subsequently was isolated. Radiolabeled PC and PG of the chloroplast and thylakoid fractions were treated with phospholipase A2 and the distribution of radiolabel between the lysophospholipid and FFA determined. Hatched bars, The fraction of radioactivity associated with the sn-1 position; black bars, the fraction of radioactivity associated with the sn-2 position. Mean values and the range from duplicate samples within one representative experiment are presented.