New evidence for the presence and function of phosphoinositides (PPIs) in the chloroplast

I would like to highlight the recent preprint by Sedaghatmehr and colleagues, where they explore the lipid composition of chloroplast membranes. Chloroplasts are specialized organelles in plant cells that are responsible for the plant’s green color and for photosynthesis. They form closed compartments inside the cell. Unlike other organelles, chloroplasts have double bilayers. Inside the outer organelle envelope, there is an internal membrane system. Inside these two membrane layers, are thylakoids that are disk-like structures, stacked on top of one another. These membranes fold into a complex network of pancake like membrane discs. There are various proteins embedded in these complex membrane structures. The proteins for transport are in the inner and outer envelope membranes. The proteins within thylakoids are almost exclusively the protein machinery which drives photosynthesis.

Lipid molecules consist of a hydrophobic tail and hydrophilic head groups. Complex lipids include extra chemical groups attached to the basic structures. One such class of lipids is the Phosphatidylinositols (PIs), which although are not very abundant, play significant functions in the cells. The inositol ring (A ring made of six carbon atoms) in the PIs has five hydroxyl groups that can be phosphorylated, and certain cellular enzymes facilitate this process. These enzymes phosphorylate three out of five hydroxyl groups to produce a bunch of new molecules called phosphatidylinositol phosphates (PPIs). Depending on number of phosphorylated groups, the PPIs are classified into mono- (PI3P, PI4P, and PI5P), bi- (PI(3,4)P₂, PI(4,5)P₂, and PI(3,5)P₂) and tri-phosphates (PI(3,4,5)P₃). The PPIs function as important signaling molecules that are recognized by proteins which alter membrane structure or regulate protein structure and function. Although, the role of PPIs in membrane organization and trafficking is well known, not much is known about the localization or signaling in plant cells.

Previous studies on PPIs have shown their involvement in chloroplast division, indicating their importance in this organelle. In this study, the authors hypothesize the presence of PPIs in the inner membrane of chloroplast. The experiments consist of PPI sensor proteins that relocate within the chloroplast when induced by stressors such as drought or heat.

Results

• Generating PPI biosensors in chloroplasts

State-of-the-art experiments that can be used to locate PPIs within plastids are limited to in vitro systems. In this preprint, the authors genetically encoded PPI sensors in plant cells. These sensors were tagged with a fluorescent dye that acts as a read-out signal. Several sensors that are specific to the different types of PPI lipids were made and introduced into the chloroplast. Some of these sensors showed a diffused fluorescence across the whole chloroplast (PI3P and PI5P) while the others were localized to specific regions, which was shown by tight puncta structures in the fluorescent images captured (PI4P, PI(4,5)P₂, and PI(3,5)P₂). The authors made sure that the detected signal was coming from inside the chloroplast and not from the outer membrane by treating the isolated organelles with enzymes that degraded proteins exposed to the chloroplast surface.

• PPI biosensors change distribution on breaking of the phosphate bond

The PPI biosensors are very specific to the type of PPIs. Enzymes that can break the phosphate bond of the PPIs change the PPI structure thus inactivating the sensors. The authors expressed more proteins that dephosphorylate the specific PPIs and observed the re-distribution of the sensors from enrichment at points to a homogenous background. This experiment validated the sensor activity and specificity.

• PPI colocalization with other proteins

Now that the authors established that the sensors accurately locate the PPIs within the chloroplast, they went on a quest to find which proteins co-localize with these important lipids. One protein of interest is named VIPP1 (short for Vesicle-inducing proteins in plastids 1). In vitro studies have shown their co-localization with PPIs in PPI-containing liposomes, but no such study has been conducted for real chloroplast systems. In this study, the authors co-expressed VIPP1 and PI3P biosensors in plant cells and verified the interactions between the protein and the PI3P lipid. They also used model lipid membrane systems (giant unilamellar vesicles, GUVs) to establish the interaction between VIPP1 and PI3P.

• Plants responding to environmental conditions

The biosensors developed in this preprint can be used to study the lipid distribution during extreme weather conditions, such as a very hot summer. The authors subjected the plants expressing the biosensors to a hot environment (40°C) for 10 days. The PPI biosensors localized inside the chloroplast showing the presence of PPIs there. This proved the involvement of the PPIs in signaling weather conditions in plants, and demonstrated how they can adapt accordingly to environmental changes.

Why I like this preprint

When it is hot outside, most animals travel to cooler areas such as shaded areas or bodies of water. Unfortunately for plants, their movement is very restricted. They must adapt to the environment accordingly which is orchestrated by a cascade of signaling molecules. The authors have addressed this issue in this preprint by suggesting the involvement of the phosphatidylinositol phosphates lipids. Since these lipids have a very low abundance but high turnover, it is very difficult to pinpoint their location. The authors have developed new biosensors that specifically attach to various PPIs giving a readout of their presence. With these sensors, it is possible to study the location of PPIs inside the cell and their interactions with other proteins. In this preprint, the authors used the sensors to show that PPIs redistribute within the cell during extreme weather conditions such as heat and drought.

Questions for the authors

• Do you think PPIs will be involved in sensing other weather conditions, such as extreme cold or floods?
• Can these biosensors be added externally to plant cells instead of just expressing them?
• What do you think will be further applications of these biosensors?

 

doi: https://doi.org/10.1242/prelights.39156

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