Figure 5. Correlative light and electron microscopy of chloroplast-to-nuclear connections induced during HR-PCD.

A. Stromules were induced by transient expression of p50-Citrine in NRIP1-Cerulean expressing N plants. Confocal z-stack images were acquired and deconvolved (left). Chloroplasts (red) have stromules (green) extending to and around the nucleus. A transmission electron micrograph of the exact same nucleus was imaged (middle). The confocal and TEM micrographs were overlaid (right) to determine the location and type of chloroplast-to-nuclear connections. An approaching stromule connection (B) and a cross-section through surrounding chloroplasts (D) were identified and examined at higher magnification and shown in panels B and D. Scale bars equal 5 μm.

B. The tips of stromules are in close contact, but the outer membrane and nuclear membrane do not directly associate. There appears to be cytosol between the stromule (S) and nucleus (N). A break or pore was observed where the tip of a stromule (S) approached the nuclear envelope (NE). Scale bar equals 0.2 μm.

C. The body of chloroplasts (Ch) often can be found in close association with nuclei (N). Numerous nuclear envelope (NE) perforations (arrowheads) were observed at the junction of the body of chloroplasts. St, starch inside the chloroplast. Scale bar equals 0.2 μm.

D. A stromule (S) wrapped around a nucleus (N) was found in nuclear groove (NG) that increased stromule contact with the nuclear envelope (NE). There were perforations (arrowheads) in the nuclear envelope of the nuclear groove. Scale bar equals 0.2 μm.

E. Oblique sections through a stromule (S)-to-nuclear (N) connection reveal numerous nuclear pores (arrows) in the nuclear envelope (NE) in close proximity to the connection. M, mitochondria near chloroplast. Scale bar equals 0.2 μm.