Potent ABA-independent activation of engineered PYL3

Abstract

Abscisic acid (ABA) plays a vital role in many developmental processes and the response to adaptive stress in plants. Under drought stress, plants enhance levels of ABA and activate ABA receptors, but under harsh environmental stress, plants usually cannot efficiently synthesize and release sufficient quantities of ABA. The response of plants to harsh environmental stress may be improved through ABA-independent activation of ABA receptors. The molecular basis of ABA-independent inhibition of group A protein phosphatases type 2C (PP2Cs) by pyrabactin resistance/Pyr1-like (PYR1/PYLs) is not yet clear. Here, we used our previously reported structures of PYL3 to first obtain the monomeric PYL3 mutant and then to introduce bulky hydrophobic residue substitutions to promote the closure of the Gate/L6/CL2 loop, thereby mimicking the conformation of ABA occupancy. Through structure-guided mutagenesis and biochemical characterization, we investigated the mechanism of ABA-independent activation of PYL3. Two types of PYL3 mutants were obtained: (a) PYL3 V108K V107L V192F can bind to ABA and effectively inhibit HAB1 without ABA; (b) PYL3 V108K V107F V192F, PYL3 V108K V107L V192F L111F and PYL3 V108K V107F V192F L111F cannot recognize ABA but can greatly inhibit HAB1 without ABA. Intriguingly, the ability of PYL3 mutants to bind to ABA was severely compromised if any two of three variable residues (V107, V192 and L111) were mutated into a bulky hydrophobic residue. The introduction of PYL3 mutants into transgenic plants will help elucidate the functionality of PYL3 in vivo and may facilitate the future production of transgenic crops with high yield and tolerance of abiotic stresses.

Keywords: ABA independent; ABA irresponsive; HAB1; PYL3; constitutive inhibition.

Fig. 1

Monomeric PYL3 mutant proteins retain the ability to inhibit HAB1 activity and bind to ABA. (A) The interactions between two apo‐PYL3 protomers were mainly mediated by F81, V108 and F188. (B) The abilities of PYL3 and its mutant proteins (1.0 μm) to inhibit HAB1 (0.5 μm) activity in the presence of ABA (5 μm) were detected by the Serine‐Threonine Phosphatase Assay System Kit. Data were expressed as mean ± standard error of the mean. (C) The heterodimer interface interactions in the PYL3–ABA–HAB1 structure were analyzed. F81 and F188 were the hydrophobic interaction centers, whereas V108 contributed little to the hydrophobic interaction. (D) EGS was used to detect the monomer or dimer state of a protein. (E) SEC assays confirmed that the point mutation of V108K or V108E could destroy the interface interactions of dimeric PYL3 and produce the monomeric forms.

Fig. 2

The site mutations around the PYL3 pocket’s entrance. (A) Superposition of apo‐PYL3 (gray cartoon, PDB: 3KLX) and PYL3‐ABA (cyan cartoon, PDB: 4DSC) structures. ABA (yellow stick) binds to the PYL3 pocket, which has hydrophobic interaction with many amino acid side chains inside and at the entrance of the PYL3 pocket. The Gate/L6/CL2 is closed, which is the most conformational change. In PYL3‐ABA, all side chains within 5 Å from ABA were displayed by cyan line or orange stick. The red dotted line represents the salt bond. (B) ABA binding to PYL3 leads to the maximum conformational change of L111 and A113 on Gate/L6/CL2. F81, V107, H139 and V192 could provide hydrophobic interaction with L111 and A113. (C) The amino acids in 14 PYLs family members involved in hydrophobic interactions, as shown in (B), were performed with sequence alignment (Fig. S2). (D) V107 and V192 were mutated into Leu or Phe with a larger hydrophobic group. (E) The L111F mutation was further introduced based on PYL3 V107L V192F. (F) The V107F mutation was additionally introduced based on PYL3 V107L V192F L111F. The number on the dotted line in (B) and (D)–(F) represents the distance (Å).

Fig. 3

The key mutations promoted the ABA‐independent inhibition of HAB1. The abilities of PYL3 and its mutant proteins to inhibit HAB1 activity in the absence of ABA were detected by the Serine‐Threonine Phosphatase Assay System Kit. The negative control was PYL3 WT protein without ABA, and the positive control was PYL3 WT in the presence of ABA. A total of 0.5 μm HAB1 and 1 µM PYL3 mutant proteins were mixed in equal volume. Data were expressed as mean ± standard error of the mean.

Fig. 4

The interactions of PYL3 and its mutant proteins with HAB1 were detected by the yeast two‐hybrid system. PYL3 and its mutant were constructed on the activation domain (AD) vector and HAB1 on the binding domain (BD) vector. Four yeast cell concentrations were selected when A _600 nm was 0.5, 0.1, 0.02 and 0.004. A total of 100 μL of 5 μm ABA solution was added to the yeast plaque center every 12 h for 2–3 days if needed.

Fig. 5

Isothermal titration calorimetric analysis of the binding of ABA to PYL3 mutants. The binding abilities of (A) PYL3 WT, (B) PYL3 V108K V107L V192F, (C) PYL3 V108K V107L V192F L111F, (D) PYL3 L111F, (E) PYL3 V108K V107F L111F and (F) PYL3 V108K V107F V192F L111F to ABA were detected by ITC.

Fig. 6

The steric hindrance between V192F, L111F or V107F mutations and ABA. (A) Structural superposition of two PYL3 protomers from PYL3–ABA (orange) and PYL3–ABA–HAB1 (PDB: 4DS8, green). (B) The residues at the pocket entrance of PYL3 WT have hydrophobic interaction with ABA. (C) V192, L111 and V107 in PYL3 were mutated into Phe. (D) There is a steric hindrance between L111F or V107F and ABA. The software PyMOL was used to measure the minimum distance from the side chain of mutated residue to ABA. (C, D) Models of PYL3 mutants.