Phospholipase Dδ negatively regulates plant thermotolerance by destabilizing cortical microtubules in Arabidopsis

Abstract

The pattern of cortical microtubule arrays plays an important role in plant growth and adaptation in response to hormonal and environmental changes. Cortical microtubules are connected with the plasma membrane (PM); however, how the membrane affects cortical microtubule organization is not well understood. Here, we showed that phospholipase Dδ (PLDδ) was associated with the PM and co-localized with microtubules in cells. In vitro analysis revealed that PLDδ bound to microtubules, resulting in microtubule disorganization. Site-specific mutations that decreased PLDδ enzymatic activity impaired its effects on destabilizing microtubule organization. Heat shock transiently activated PLDδ, without any change of its PM localization, triggering microtubule dissociation from PM and depolymerization and seedling death in Arabidopsis, but these effects were alleviated in pldδ knockout mutants. Complementation of pldδ with wild-type PLDδ, but not mutated PLDδ, restored the phenotypes of microtubules and seedling survival to those of wild-type Arabidopsis. Thus, we conclude that the PM-associated PLDδ negatively regulates plant thermotolerance via destabilizing cortical microtubules, in an activity-dependent manner, rather than its subcellular translocation.

Phosphatidic Acid Directly Regulates PINOID-Dependent Phosphorylation and Activation of the PIN-FORMED2 Auxin Efflux Transporter in Response to Salt Stress

“…In Arabidopsis, PLDa and PLDd are the most abundant PLDs, and their activities increase upon exposure to salt stress (Bargmann et al, 2009;Yu et al, 2010;Zhang et al, 2012b). The absence of PLDs in plda1, pldd-1, and pldd-2 single mutants and in plda1 pldd-1 and plda1 pldd-2 double mutants was verified by immunoblotting using anti-PLDa1 and anti-PLDd antibodies ( Figure 1A; Guo et al, 2012;Zhang et al, 2012bZhang et al, , 2017. Deletion of PLDa1 or PLDd decreased total PLD (PLDa1 and PLDd) activity, and PLD activity was decreased markedly in the plda1 pldd-1 and plda1 pldd-2 double mutants ( Figure 1B).…”

“…The homozygous lines were confirmed by PCR. The homozygous pld mutants (plda1, pldd-1, pldd-2, and pldz2) and wag2 (SALK_070240) were described previously (Li et al, 2006;Zhang et al, 2012bZhang et al, , 2017Miao et al, 2018). The pid mutant is sterile and therefore must be propagated as a heterozygote.…”

“…Approximately 40% of homozygous pid mutants develop three symmetrically arranged cotyledons, and selecting three cotyledons serves as an effective screen for homozygous pid seedlings during their further phenotypical analysis, and the heterozygous lines were used for complementation experiments. The plda1 pldd-1 and plda1 pldd-2 double mutants were generated by crossing and verified by immunoblotting using anti-PLDa1 and anti-PLDd antibodies (Zhang et al, 2012b(Zhang et al, , 2017, respectively. The Arabidopsis lines Pro PIN2 :PIN2-GFP and DII-VENUS have been described previously (Blilou et al, 2005;Xu and Scheres, 2005;Brunoud et al, 2012).…”

“…Total proteins from 6-d-old Arabidopsis seedlings were isolated and immunoblotted using an anti-PLDa1 or anti-PLDd antibody (Zhang et al, 2012b(Zhang et al, , 2017 and incubated with a secondary antibody conjugated to alkaline phosphatase (Sigma-Aldrich; catalog No. SAB3700957).…”

Update: Plant Cortical Microtubule Arrays

“…However, conflicting results were obtained in a later study, which found that cortical microtubule remained attached to the plasma membrane of tobacco BY-2 protoplasts after treatment with n-butanol (Hirase et al, 2006). Recently, heat shock was found to activate PLD, in which microtubules dissociated from the plasma membrane and became destabilized, leading to the disorganization of cortical arrays (Zhang et al, 2017). In addition to PLD, cortical microtubules partially detach from the membrane and exhibit extensive waving in clasp-1 cells due to cytoplasmic streaming before reattaching to the membrane (Ambrose and Wasteneys, 2008).…”