Abstract
Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a novel signaling mechanism, especially for participation of chloroplast proteins (e.g. transcription factors) in retrograde signaling, thereby offering new opportunities to regulate pathways outside chloroplasts.
Highlights
Chloroplasts support life on earth by performing photosynthesis
Two new chloroplast transformation vectors were designed for expressing PTD-green fluorescent protein (GFP) in lettuce or tobacco (Figure 1A and 1B) and GFP-RC101 vector from a previous study was used [15]
The design of chloroplast transformation vectors used here is similar to previous studies in our lab [15,20]
Summary
Chloroplasts support life on earth by performing photosynthesis. In addition to carbohydrates, plastids synthesize amino acids, proteins, fatty acids, pigments, hormones, vitamins and therapeutic biomolecules. Coordination and assembly of multi-subunit complexes or biosynthetic pathways encoded by the plastid and nuclear genome requires efficient and accurate signaling between these two cellular compartments. The anterograde signaling pathways, in which the nucleus encodes plastid protein subunits, transcription factors and RNA binding proteins to coordinate plastid functions [1], have been studied in depth. It has been known for several decades that nuclear gene expression is regulated by plastids via retrograde signaling, the molecular mechanism is still unknown [2]. PTM, a chloroplast envelope-bound plant homeodomain transcription factor, has been shown to be involved in retrograde signal pathways [9]. It is likely that retrograde signals from plastids during development (greening) are different from those generated under stress and might involve transcripts, proteins or other catalytic biomolecules
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