Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs

Abstract

SUMMARYAbscisic acid (ABA) is a key phytohormone involved in adaption to environmental stress and regulation of plant development. Clade A protein phosphatases type 2C (PP2Cs), such as HAB1, are key negative regulators of ABA signaling in Arabidopsis. To obtain further insight into regulation of HAB1 function by ABA, we have screened for HAB1-interacting partners using a yeast two-hybrid approach. Three proteins were identified, PYL5, PYL6 and PYL8, which belong to a 14-member subfamily of the Bet v1-like superfamily. HAB1-PYL5 interaction was confirmed using BiFC and co-immunoprecipitation assays. PYL5 over-expression led to a globally enhanced response to ABA, in contrast to the opposite phenotype reported for HAB1-over-expressing plants. F 2 plants that over-expressed both HAB1 and PYL5 showed an enhanced response to ABA, indicating that PYL5 antagonizes HAB1 function. PYL5 and other members of its protein family inhibited HAB1, ABI1 and ABI2 phosphatase activity in an ABA-dependent manner. Isothermal titration calorimetry revealed saturable binding of (+)ABA to PYL5, with K d values of 1.1 lM or 38 nM in the absence or presence of the PP2C catalytic core of HAB1, respectively. Our work indicates that PYL5 is a cytosolic and nuclear ABA receptor that activates ABA signaling through direct inhibition of clade A PP2Cs. Moreover, we show that enhanced resistance to drought can be obtained through PYL5-mediated inhibition of clade A PP2Cs.

ABA signalling is fine-tuned by antagonistic HAB1 variants

“…The last several years have been very fruitful in terms of elucidating the molecular mechanism of PYL-controlled PP2C activity 5,[11][12][13][14] . However, until now, no study has investigated the mechanisms underlying PP2C activity regulation at the post-transcriptional level.…”

“…In contrast, the binding of HAB1 by PYLs at the same ratio and in the presence of ABA turns ABA signalling on [5][6][7] . However, it has long been noticed that in the presence of ABA, PYL expression is decreased, while HAB1 and SnRK2.6 expression is greatly increased 14 . Taking the ratios of PYLs and HAB1 or HAB1 and SnRK2.6 into account, the decrease in PYLs and increases in HAB1 and SnRK2.6 under prolonged ABA treatment would promote the interaction of HAB1 and SnRK2.6 and rapidly switch ABA signalling off.…”

“…In Arabidopsis, nine group A PP2Cs interact with PYLs in the presence of ABA 5,[11][12][13][14] . HAB1 is among the best-characterized PP2Cs in ABA signalling 15 .…”

Structural insights into PYR/PYL/RCAR ABA receptors and PP2Cs

“…Indeed, binding of (-)ABA to the PYL5 receptor has been measured using isothermal titration calorimetry [10], although the Kd for the natural (+)enantiomer was 20-fold lower (1.1 versus 19.1 M), which indicates a higher affinity for ABA binding of the natural form. Additionally, (-)ABA promoted interaction of PYL2, PYL3 and PYL4 with HAB1 [8].…”

“…Finally, a chemical genetic approach using a synthetic selective ABA agonist, pyrabactin, made it possible to identify a family of soluble ABA receptors, named PYR/PYL/RCAR for PYRABACTIN RESISTANCE1/PYR1-LIKE/ REGULATORY COMPONENTS OF ABA RECEPTORS, respectively [8,9]. In a convergent approach, yeast-two hybrid screenings [9,10] and in planta interaction studies [11] identified these receptors through their capacity to bind to phosphatases type-2C (PP2Cs), which are key negative regulatory components of the pathway involved in the dephosphorylation of certain sucrose non-fermenting 1-related subfamily 2 (SnRK2) kinases [12,13,14].…”