Figure 1.

RSH3-GFP Overexpression Reduces Chloroplast Function.

(A) OX:RSH3-GFP.1 (OX:RSH3^GFP) and OX:RSH2-GFP.1 (OX:RSH2^GFP) plants are small and pale (above) and have a high basal chlorophyll fluorescence, F0 (below). Plants shown were grown on plates for 16 DAS. F0 false-color scale bar, 50 to 350 arbitrary units.

(B) OX:RSH3GFP.1 seedlings have significantly lower chlorophyll levels and chlorophyll a/b ratios than the wild type (n = 4 plants, 12 DAS).

(C) Immunoblots on equal quantities or dilutions of total protein from wild-type seedlings and seedlings overexpressing RSH3-GFP 12 DAS using the indicated antibodies against signature chloroplast proteins. Chloroplast-encoded proteins are indicated by green text. RBCL was revealed by Coomassie Brilliant Blue staining (CBB).

(D) Total RNA from wild-type plants and RSH3-GFP-overexpressing plants showing cytosolic (black) and chloroplastic rRNA (green).

(E) ppGpp was extracted from the leaves of soil-grown plants 32 DAS and quantified by ultraperformance liquid chromatography-mass spectrometry; P = 0.00013, n = 3 biological replicates.

(F) F0 images of wild-type plants, OX:RSH3-GFP.1 plants, and OX:RSH3-GFP.1 plants crossed with inducible MESH plants 12 DAS. Plants were grown on medium containing the carrier (DMSO) or 1 µM dexamethasone (DEX). MESH, catalytically active chloroplastic enzyme; ΔMESH, catalytically inactive chloroplastic enzyme; cytMESH, an active MESH targeted to the cytoplasm. cytMESH plants were segregating for OX:RSH3-GFP.1.

(G) Immunoblots showing the accumulation of RSH3-GFP and MESH proteins in total extracts from the same plants as analyzed for F0 in (F). For cytMESH, only plants overexpressing RSH3-GFP were selected for protein extraction. PR, Ponceau Red. Significance was tested using the two-way Student’s t test, ** P < 0.01. Error bars indicate se.