New lipid biosensor shines some light on stress response

New lipid biosensor shines some light on stress response

PA (phosphatidic acid) is a membrane lipid, however it is also an important signalling lipid involved in the regulation of important cellular processes. In plants PA has been shown to be involved in the regulation of stomata opening as well as in drought and salt stress. PA can be made through a number of pathways, of which the phospholipase D (PLD)-mediated hydrolysis of phospholipids is one. PA signalling takes place through direct interaction with effector proteins which affects their activity. While it is known that PA shows dynamic changes in response to different stimuli, up to now we have been unable to observed these directly.

In the recent paper of Li et al, they report the development of a new type of biosensor for PA, which enables the measurements of change in concentration and dynamics of bioactive PA. Using this biosensor they were able to show that not only PLD generates bioactive PA in response to ABA, salt and osmotic stress, but that the dynamics with which PA is generated is dependent on the type of stimuli observed. Moreover, they showed that binding of the PA biosensor was pH dependent together with that there is a pH dependent element in plant salt tolerance.

To make this new type of ratiometric PA-biosensor the authors made use of the PA-binding domain of  the protein RHOHD. First they showed that this domain only binds PA and no other lipids. For making the biosensor ratiometric the authors made use of FRET, by placing the PA-binding domain between two fluorescent proteins, with suitable linkers they created a biosensor that upon binding PA had a greater FRET efficiency. This enabled them to calculate the ratio between bound and unbound PA-biosensor. To be able to measure quick changes the authors added a plasma membrane localization signal to the biosensor. Testing the biosensor in Arabidopsis using external application of PA they showed that the biosensor had a quantitative response to PA.

Now they had a ratiometric PA-biosensor the authors wanted to test it with known inducers of PA. First they tested how the response was to externally applied ABA. Using the biosensor they showed that ABA caused an increase in FRET signal, and thus PA, within 200 seconds. As PA can be synthesised via different routes they tested if the enzyme PLD has a role in the production of PA in response of ABA. Using n-butanol to suppress PA production by PLD, they showed that the increase of PA in response to ABA was reduced. The use of a pld mutant line also showed a reduced and delayed increase of PA in response to ABA. Suggesting that PA production in response to ABA is primarily done via PLD.

Next they tested response to salt stress. Upon application of NaCl to the root tip it almost immediately caused an increase in FRET signal, this was most strongly seen in the root tip, but also observed in the differentiation and maturation zone of the root. Again they tested if PA in response to sat stress was made via PLD, by applying n-butanol and using the pld mutant. Showing that indeed PLD is responsible for the production of a large amount of PA in response to salt stress. While the pld mutant shows a reduction and delayed increase of PA in response to salt compared to wild-type, this reduction and delay in response was less than seen for the PA response to ABA in the pld mutant.

As salt stress has both an ionic and an osmotic component, the authors tested also the effect of osmotic stress by application of mannitol. Again upon application of mannitol an increase in FRET signal was observed.  However in the pld mutant the FRET signal in response to mannitol was severely delayed and reduced. With this the authors concluded that while ABA, salt and osmotic stress al resulted in an increase in PA derived from PLD, the dynamics of this PA increase is dependent on the type of stress perceived.

Lastly the authors reasoned that the binding of the biosensor to PA was pH dependent. To test this the authors analysed the binding of the biosensor to PA under neutral (5.8), low (4.5) and high (7.0) pH. Whit this they showed that the binding of the biosensor to PA was reduced when the roots were placed from neutral into low pH solution, and that placing them back at neutral pH solution restored the binding to PA. Moreover, transferring the roots form neutral in to high pH solution resulted in an increase in binding to PA, and placing the roots back at a neutral pH solution reduced the binding to PA to the original levels. Repeating this last analysis in the presence of salt the authors showed that while salt still elicited a PA response, the binding of the biosensor was affected by the pH in which the roots were placed with a stronger signal at a higher pH level, which was reduced when the pH was lowered. Showing that the ability to respond to salt stress has a pH-dependent element.

With this Li et al., not developed an very usefull new ratiometric PA-biosensor, but also shown us what we have been missing in the previous studies of PA response to ABA and salt stress.

When NaCl knocks on plant’s door

When NaCl knocks on plant’s door

When a plant root comes across a patch of soil with a higher amount of salt, one of the first things it does is sending up a wave of Ca2+ signal up to the shoot. Depending of the size of the plant this Ca2+ wave can reach the top within minutes. There it will regulate gene expression and protein activity to prepare the shoot for the osmotic and salt stress. For example activating the expression of genes that encode Na+ channels that will either exclude Na+ from the cell or compartmentalise it.

As plants don’t have a nervous system like animals do, signals between distant part of the plant need to travel from cell to cell, in general this is a relatively slow process. However some signals travel faster than others. Ca2+ are versatile second messengers, and in contrast to some other signalling molecules, Ca2+ has been shown to be able to travel quickly from cell to cell. In general the Ca2+ concentration in the cytoplasm is relatively low, however cells have high levels of Ca2+ stored in storage compartments. Upon a stimulus this stored Ca2+ will be released to increase the cytoplasmic Ca2+ levels which in turn will activate a set of proteins that sent a signal out of the cell that is picked up by neighbouring cells, setting off the whole signalling cascade again.

So what will happen when the root comes across a patch of high saline soil. The first thing it will notice that there is an increase in Na+ ions. These Na+ ions will bind to a negatively charged membrane lipid called glycosyl inositol phosphorylceramide or GIPC for short. The binding of Na+ to GIPC then activates an GIPC-associated Ca2+ channel. Resulting in an influx of Ca2+ ions into the cytosol. One of the things these newly entered Ca2+ ions will trigger is the signalling cascade triggering the Ca2+ wave towards distal parts of the plant, telling the rest of the plant to prepare for an influx of Na+ and Cl– ions.

The best model to date indicates that to create this Ca2+ wave the plant makes use of H2O2 which can feely diffuse between cells. Upon entering the cell Ca2+ is detected by CPKs and CBL-CIPKs which in turn phosphorylate a plasma membrane bound RBOH NADPH oxidase. The phosphorylation of RBOH results in its activation and the production of H2O2, which will then diffuse to the neighbouring cells. There it will trigger Ca2+ release from the vacuole by either directly or indirectly activating the vacuolar Two-Pore Channel 1 or TPC1. This newly released Ca2+ will again result in the activation of RBOH. Resulting in a self-sustaining cell-to cell-propagating long-distance Ca2+ wave.

ca2+ wave corrected
Model of the Ca2+ wave signaling cascade, showing that it keeps going once it started.

While researchers are still trying to get the details right and verify this model beyond any doubt, it fits nicely with our current evidence. Part of the difficulty is that plants have many CPKs, CBL-CIPKs and RBOHs which in addition forming the Ca2+ wave are involved in many Ca2+ and ROS signalling pathways. making deciphering their interactions and effects complex and time consuming.

References

Jiang et al., Nature, 2019

Steinhorst and Kudla, Nature 2019

Steinhorst and Kudla, Current opinion in plant biology, 2014, 22:14-21

Choi et al., PNAS, 2014 111:6497-6502

Stephan and Schroeder, PNAS, 2014, 111:6126-6127

Koster et al., Plant Biology, 2019, 21:39-48

The sensing of salt

The sensing of salt

How  plants sense salt has at last been deciphered by the group of Zhen-Ming Pei. For their last publication in Nature they went on the hunt for mutants that did not show salt induced Ca2+ spikes. This resulted in the identification of a mutant they named moca1 for monocation-induced [Ca2+]i increases 1.  moca1 is hypersensitive to salt stress. One of the reasons is that, unlike wildtype plants who through activation of the SOS-pathway try to actively export Na+ from the cell,  moca1 plants don’t do this.

They found that moca1 had a mutation in a gene encoding for an enzyme in the pathway of the biosynthesis of membrane lipid called glycosyl inositol phosphorylceramide or GIPC for short. GIPCs are located at the outside of the plasma membrane and have a negative charge. Analysing the levels of GIPC in moca1 plants showed that they were reduced. However, when grown under optimal conditions it was not possible to distinguish moca1 from wildtype, only under salt stress conditions moca1 showed a phenotype. This suggested that while the levels of GIPC in moca1 plants were low, they were high enough to survive under normal conditions. But that under salt stress conditions higher levels of GIPC are needed.

Being puzzled by this, the researches subsequently analysed how GIPC could play a role in salt-induced increases of Ca2+ in the cell. Revealing that in the abcense of GIPC the cell-surface potential is not affected by NaCl, while NaCl does have an effect on the viability of the cells. Furthermore, they show that Na+ bind to GIPC outside the cell. The combination of these results they link to the opening of Ca2+ channels needed for the observed Ca2+ influx upon NaCl perception. Although, upto date it is not exactly know how the opening of Ca2+ channels in plants is regulated.

In the accompanying review of this article an alternative model was suggested that the binding of Na+ to GIPC would result in the formation of a microdomain in the plasma membrane. This microdomain would contain in addition to GIPC, Ca2+ channels also signalling proteins. In the alternative model it was proposed that some of these signalling proteins then activate the Ca2+ channel.

While it is possible that the binding of Na+ to GIPC leads to microdomain formation,  it adds one extra step to the process.  And as Occam’s razor reminds us, the simplest option is often the best.  Therefore, if there needs to be an alternative model to that of the authors mine would be that under non-stress conditions GIPC is bound to Ca2+ channels, thereby inactivating them. However when Na+ levels rise Na+ outcompetes the Ca2+ channels for GIPC binding. Releasing the inhibition from the Ca2+ channels, allowing them to open.

Whatever model turns out to be true, the groups of James Siedow and Zhen-Ming Pei have done great work to show us how Na+ is perceived by the plant and that disruption of this perception results in an absence of the Ca2+ signal needed to alert the rest of the plant that it is under salt stress.

Great talk about Cross-kingdom RNAi by Hailing Jin

Great talk about Cross-kingdom RNAi by Hailing Jin

Last week we were treated by a fantastic seminar from Hailing Jin from the University of California. In the over-airconditioned seminar room we quickly forgot about the cold while she introduced us in the world of cross-kingdom RNAi and small RNA trafficking between plants and fungal pathogens. She started with telling us how small RNAs are formed from their precursors using Dicer-like (DCL) proteins and how these small RNAs are incorporated into AGO proteins to induce gene silencing, which we call RNA interference (RNAi). And how RNAi is a conserved regulatory mechanism across almost all eukaryotic cellular processes. We then were introduced to her pathogen of choice in her study of plant-fungal pathogen interaction, Botrytis cinerea (Bc). Bc is actually around us in the air and is probably the one you see when your fruit of veg has gone mouldy. However, the main reason she used them is that the have their own small RNA machinery and can thus process small RNAs by themselves. Part of the way how Bc infects plants is that after penetrating the plant cell, Bc secretes small RNAs into the plant cell. These Bc-small RNAs are then loaded by the plant onto its own AGO machinery, which then will silence the genes targeted by the Bc-small RNA. The result of this is an inhibition of the plant immune response, enhancing the virulence of Bc. Subsequent research by her and other groups has shown that similar mechanisms are used by fungi infecting mammalian cells, as well as by parasitic plants and bacteria and their interaction with their host.

Hailing Jin told us then the realisation that if small RNAs can travel from fungi to plant, they supposedly should be able to travel into the other direction as well. The trouble with this was that in order to be able to show that an organism has received small RNAs from another organism you need to be able to separate the two organisms from each other, before the isolation of RNA. As both plants and fungi have cell walls, which actually consist of different compositions, the separation should be possible using sequential protoplast purification. So she convinced a postdoc to give it a try or actually “Give it three tries, as when you try something new you might fail the first time, but with troubleshooting you will succeed the second or third time.” Therefore, with some tries a postdoc from her lab was able to do this by isolating Bc-protoplast from infected leaves. Subsequent analysis of the small RNAs of these Bc-protoplasts showed that not only they contained small RNAs originating from plants, but also that these small RNAs where actively transported to the fungal cells.

In the following quest to find out how these plant-small RNAs were delivered to the fungi Hailing Jin looked into the possible delivery options that are known. One of these are extracellular vesicles, which have been implicated in the transport of small RNAs in mammalian cells. In order to find out if in plants these extracellular vesicles contained small RNAs they collected the extracellular fluid from the phloem from both control and Bc infected leaves. This showed not only that these extracellular vesicles contained small RNAs, but also that when coming from Bc-infected leaves that these small RNAs matched those found in Bc-protoplast from infected leaves. They then went on to confirm that these extracellular vesicles are exosomes, by showing that the plant exosome markers TETRASPANIN (TET), TET8 and TET9 are induced by Bc infection. Using TET8-GFP, they show that TET8-GFP accumulates at the infection site, were TET8-GFP labelled vesicles are secreted. Subsequent analysis of the uptake of these vesicles showed that they are readily taken up by Bc. To confirm that TET8 and TET9 are required for the vesicles transporting the small RNAs they created plants with a reduced expression of TET8 and TET9, and infected these plants with Bc. Not only were these plants now more susceptible to Bc infection, they also had a reduced transfer of plant-small RNAs to Bc. Indicating that the small RNAs that the plant transfers to Bc contribute to its defence against Bc. They then looked into which genes the plant-small RNAs targets in Bc. It turned out that a lot of these genes were actually involved in vesicle trafficking.

Haining Jin went then on to discuss that small RNA trafficking is a level of communication across-species, and how we can use this for plant protection from fungal pathogens. As this is an interesting topic that deserves a post on its own I will talk about that in more detail later. But I will finish with giving you the links to  Haining Jin’s articles including those discussing how cross-kingdom RNAi can be used for plant protection.

References

Weiberg et al., Science, 342:118-23, 2013
Cai et al., Science 360:1126-29, 2018
Weiberg et al., Current Opinion in Biotechnology, 32:207-215 2015
Huang et al., Cell Host and Microbe, 26: 173-182, 2019
Wang et al., Nature Plants 2:16151, 2016