Arabidopsis thaliana is the lab rat of plant sciences. Mutants tweaked in certain genes can show the effects of hormones on plants, but it can be hard to change one response without changing some others.
How do plants work? A common method to find out what the various things inside a plant do is to get a mutant and test it against a known plant to see what the mutant effects are. Arabidopsis thaliana is the go-to plant for this kind of work. It has a small, well-known genome. It also has a large number of people working on it, so it’s possible to compare results.
Tetiana Kalachova, Martin Janda and colleagues have been working with a particular Arabidopsis mutant, pi4kβ1β2. This mutant accumulates salicylic acid, a key component of the painkiller aspirin. Plants don’t get headaches, but they do get stress, and salicylic acid is an essential hormone for alleviating stress. The team have already found that the pi4kβ1β2 mutant has stunted growth, deposition of secondary metabolites in leaves, as resistance to several pathogens. But working out precisely what the pi4kβ1β2 mutation is doing is challenging, as author Prof Ruelland explained. “If we look directly at the mutant, we are likely to see the effect of high SA accumulation. The effect of the mutations not dependent on the high SA may be masked.”
Pulling apart what the mutation is doing meant the authors we able to look at some other effects of the changes. Prof Ruelland said, “we are interested in understanding the roles of the phosphatidylinositol 4-kinase beta1 and 2. They are enzymes involved in the synthesis of phosphoinositides. They are involved in processes such as trafficking, signalling, etc.. it is important to be able to identify the processes controlled by these enzymes.”
To find out what pi4kβ1β2 did without salicylic acid, the authors crossed a pi4kβ1β2 mutant with a sid2 mutant. sid2 is a useful plant because it’s poor at producing salicylic acid. So the offspring of these two plants should have the effects of pi4kβ1β2 except for the benefits of more salicylic acid. This preparation, Prof Ruelland said, required months of work to get plants to start the study. “A creation of multiple mutants (carrying several mutations at once) is a multistage process. Arabidopsis life cycle is quite short, so the whole “creation process” for triple mutant requires 3-4 full generations or 6-8 month (if all goes smoothly). At first, you have to fecundate one of mutant of interest with the pollen of another. It is delicate work, but doable after some training. Then you observe a hybrid silique growing, collect seeds and sow them. It is generation F1. Once F1 plants develop leaves, it becomes possible to sample them for genotyping. If crossing was successful ( = F1 plants are heterozygous for the mutations of interest), several individual plants are left for self-pollination to obtain F2 seeds. F2 plants are then grown and genotyped, searching now for homozygous individuals.”
“If mutations of interest are independent (i.e. genes are located at different chromosomes, or at the same chromosome but at a reasonably big distance to allow independent inheritance), then the chance to get homozygous plants in F2 for each mutation will be 1/4. Due to Mendelian genetics, for a double mutant, this chance will be 1/16, and for a triple mutant 1/64. Each additional mutation multiplies the possible combinations of alleles and so makes the segregation process longer. The process gets more complicated if one mutation (or their coincidence) strongly affects growth or fertility.”
The authors tested the plants’ responses to pathogens and compared them to the parent plants and wild-type arabidopsis. The results show the importance of salicylic acid in managing defences against infection through cross-talk with other plant hormones. One of the puzzling results was that “both callose accumulation and fungal penetration were enhanced in the pi4kβ1β2 double mutant compared to wild-type plants.” This result might seem odd as callose is a defence the plant uses to fight fungal infection, so how did both measurements rise? Prof Ruelland says it’s about looking how callose works in the plant.
“Callose is usually deposited in the cell wall during infection or damage. However, its role is still discussed. It can be both a physical barrier but also signalling to the neighbouring cells. In the case of fungal penetration, forming papilae and then hyphae are making invaginations into plant cells, and this “place of contact” gets reinforced by callose. Also, callose is stocked around non-attacked cells, those that recognize fungal presence by chemical markers.”
“In both sid2pi4kβ1β2 and pi4kβ1β2 mutants, callose deposition is generally misregulated: they overproduce callose in response to stimulation. However, this was not sufficient to stop pathogen penetration. Another explanation can be that more callose in mutants is just a marker of bigger pathogen success. The reason why the absence of PI4K made plants susceptible to non-host fungus is though still unknown.”
The role of salicylic acid is a topic that will reward further study, as Prof Ruelland said. “Important hormones like abscisic acid derivatives and auxin conjugates are controlled by high SA. We would like to know more about how SA controls the levels of these hormones.”
The research will have practical value outside the lab for plant breeders. Prof Ruelland said, “We generally aim to understand how the plant immune system works, to further create resistant crop varieties. Indeed, manipulations with productivity traits often compromise defence and can even make plants susceptible to unusual pathogens. We believe that phospholipid signalling machinery is a target for modification to improve plant basal immunity and protect against fast-evolving pathogens.”
Kalachova, T., Janda, M., Šašek, V., Ortmannová, J., Nováková, P., Dobrev, I. P., … Ruelland, E. (2019). Identification of salicylic acid-independent responses in an Arabidopsis phosphatidylinositol 4-kinase beta double mutant. Annals of Botany. https://doi.org/10.1093/aob/mcz112