New lipid biosensor shines some light on stress response

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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.