Three-dimensional organization of higher-plant chloroplast thylakoid membranes revealed by electron tomography

Abstract

The light-harvesting and energy-transducing functions of the chloroplast are performed within an intricate lamellar system of membranes, called thylakoid membranes, which are differentiated into granum and stroma lamellar domains. Using dual-axis electron microscope tomography, we determined the three-dimensional organization of the chloroplast thylakoid membranes within cryo-immobilized, freeze-substituted lettuce (Lactuca sativa) leaves. We found that the grana are built of repeating units that consist of paired layers formed by bifurcations of stroma lamellar sheets, which fuse within the granum body. These units are rotated relative to each other around the axis of the granum cylinder. One of the layers that makes up the pair bends upwards at its edge and fuses with the layer above it, whereas the other layer bends in the opposite direction and merges with the layer below. As a result, each unit in the granum is directly connected to its neighbors as well as to the surrounding stroma lamellae. This highly connected morphology has important consequences for the formation and function of the thylakoid membranes as well as for their stacking/unstacking response to variations in light conditions.

Figure 1.

Tomographic Sections of a Chloroplast. (A) A low magnification overview. Grana (G) are interconnected by multiple stroma thylakoids (SL). The latter also make rare connections with the inner envelope membrane (EM) of the chloroplast (inset). Ribosomes appear as heavily stained particles; PG denotes plastoglobuli. This and the following panels represent ∼20-nm-thick sections cut through the tomographic volume. A complete set of the tomographic slices used to generate the volume is provided in Supplemental Movie 1 online. (B) A close-up of a granum-stroma assembly. The stroma lamellae, shown in different colors, intersect the granum body in multiple, approximately parallel, planes. Bifurcation of some of the stromal membranes into adjacent granum layers is clearly observed. Note that the top- and bottom-most layers of the granum are smooth, in contrast with the other layers of the stack, which are highly granulated. (C) Tomographic slice of a granum-stroma lamellae ensemble taken in a direction roughly parallel to the plane of the grana layers. The section was made at the level of the stroma membrane marked in yellow in (B). The other stroma lamellae that surround the coin-shaped granum (dashed line) appear as ribbons rather than contiguous layers because they ripple in and out of the section plane. The granum surface appears grainy because the section was taken through the body of the granum rather than at its ends. At this angle, the four thick serial sections used in the study are apparent. Bars = 100 nm.

Figure 2.

3D Organization of a Granum-Stroma Assembly. The structure was generated from the tomographic data shown in Figure 1. (A) The granum layers are contiguous with the stroma membranes that bifurcate at the granum-stroma interface. Internal connections between adjacent layers are indicated by an arrow and an arrowhead. G, grana; SL, stroma thylakoids. (B) and (C) To better visualize the connectivity of the assembly, the structure shown in (A) was enlarged, clipped, rotated by ∼20° (B) and 40° (C), and the upper layer of the stack has been removed. Connections between adjacent layers that are not marked by an arrow or an arrowhead are bifurcations of the stroma lamellae. In all panels, the structure was expanded along the z axis to provide a clear view of the interior of the granum.

Figure 3.

A Topological Model of the Granum. The granum is made of repeating units, each consisting of two layers (red and yellow), which are formed by bifurcations of the stroma lamellae (gray). In each unit, part of the top layer (red) bends upward and fuses with the layer above it, whereas the other layer (yellow) bends downward at the opposite side and fuses with the layer below. As indicated by the blue dashed lines, going from one unit to the one above it involves a counterclockwise rotation of ∼25° around the axis of the granum cylinder. Note that the spacing between the three units that constitute the stack has been grossly exaggerated for clarity and that the bent regions that interconnect them appear to take a significant part of the area of the granum layers. In reality, all the layers in the stack are closely appressed and run parallel to each other along almost their entire area; bending occurs only at the edge of the layers.

Figure 4.

Thylakoid Network. Architecture of an ensemble consisting of two grana interconnected by multiple stroma lamellae. The figures shown in (B) and (C) are rotated views of the structure shown in (A), with two (B) or four (C) of the lamellae removed. The stroma lamellae form wide slightly curved sheets that run parallel to each other and intersect the grana at an angle that is roughly perpendicular to the axis of the granum cylinder. The grana (surface-rendered gray objects) act as defined regions of sheet consolidation and increased connectivity. The arrows in (C) define the plane of the layers inside the granum.