PYL1- and PYL8-like ABA Receptors of Nicotiana benthamiana Play a Key Role in ABA Response in Seed and Vegetative Tissue

Abstract

To face the challenges of climate change and sustainable food production, it is essential to develop crop genome editing techniques to pinpoint key genes involved in abiotic stress signaling. The identification of those prevailing abscisic acid (ABA) receptors that mediate plant-environment interactions is quite challenging in polyploid plants because of the high number of genes in the PYR/PYL/RCAR ABA receptor family. Nicotiana benthamiana is a biotechnological crop amenable to genome editing, and given the importance of ABA signaling in coping with drought stress, we initiated the analysis of its 23-member family of ABA receptors through multiplex CRISPR/Cas9-mediated editing. We generated several high-order mutants impaired in NbPYL1-like and NbPYL8-like receptors, which showed certain insensitivity to ABA for inhibition of seedling establishment, growth, and development of shoot and lateral roots as well as reduced sensitivity to the PYL1-agonist cyanabactin (CB). However, in these high-order mutants, regulation of transpiration was not affected and was responsive to ABA treatment. This reveals a robust and redundant control of transpiration in this allotetraploid plant that probably reflects its origin from the extreme habitat of central Australia.

Keywords: ABA receptors; ABA sensitivity; CRISPR/Cas9; Nicotiana benthamiana; biotechnological crop; drought; extremophile; gene editing; multiplex mutations; tetraploid.

Figure 1

Location of the ABA receptor genes in the chromosomes of Nicotiana benthamiana (n = 19). The size (Mbp) of each chromosome is indicated in red and shown by its relative length. The position of each ABA receptor gene is indicated by bars. Those genes labeled in grey indicate sequences that show homology to ABA receptors, but whose gene products likely lead to non-functional receptors because of severe truncations or the presence of nonsense mutations. The figure was generated using the genome coordinates indicated in Table S1 according to www.nbenth.com (v3.3) (accessed on 30 November 2021).

Figure 2

Phylogenetic analysis of PYL proteins in Arabidopsis thaliana and Nicotiana benthamiana. A total of 14 AtPYLs (PYR1 to PYL13) and 23 NbPYLs were included to generate the cladogram by using the MegAlign 7.0 software. The NbPYL proteins were clustered in three subfamilies as previously described for PYLs in other plant species [24], including subfamily III of putative dimeric receptors. NbPYLs orthologous to AtPYL11-13 were not detected. Red asterisks mark the genes described in this work. An alphanumeric descriptor has been included for each PYL receptor to facilitate naming.

Figure 3

Relative expression of NbPYLs in the fifth leaf and scheme of CRISPR/Cas9 guide RNAs (gRNAs) used in this study. (A) The gene expression of NbPYLs was determined by RNA-seq analysis. Data for mRNA expression of ABA receptors in leaves was normalized and expressed in FPKM (Fragments per kilobase of exon model per million mapped reads). Target genes selected for CRISPR/Cas9-mediated editing are indicated by red asterisks, including the number (see below Figure 3B) of the gRNAs designed for each receptor gene. (B) Structure of the polycistronic transcriptional units in the GoldenBraid-based plasmid construct indicating the position of each gRNA after the U6-26 promoter. The last position (indicated in red) of each transcriptional unit turned out to be the most effective for gene editing. The efficiency of generating edited alleles is indicated below as the percentage of mutations found after sequencing of T1 plants. Given the high degree of identity between PYL1a and PYL1b, or PYL8a and PYL8b, the gRNAs 1 and 6, or 2 and 7, served to target both pairs of genes, respectively.

Figure 4

Amino acid sequence alignment of AtPYL1, AtPYR1, AtPYL8, and five NbPYLs. (A) Sequence and secondary structure alignment of ABA receptors are indicated. The secondary structure of the NbPYLs is predicted according to the crystallographic structure of AtPYL1 (Protein DataBank Code 3JRS), and was generated using the ESPRIPT program (http://espript.ibcp.fr/ESPript/ESPript/ (accessed on 16 January 2022)). Boxes indicate the position of the gate and latch loops. Blue asterisks mark residues involved in interactions with ABA. The dashed line indicates the 8 amino acid residues that are predicted to be deleted in the pyl1a-2 allele. (B) Purified recombinant His-tagged NbPYL1b shows ABA-dependent inhibition of the PP2C HAB1. Left panel, CB- and ABA-dependent inhibition of the PP2C HAB1 assayed using pNPP as a substrate. Right panel, determination of the IC50 for inhibition of HAB1 by CB or ABA and NbPYL1b. (C) The elution profile of NbPYL1b after gel filtration chromatography indicates that the protein was eluted as a dimer. The positions of the molecular weight markers are indicated. The inset represents the calibration curve for the column, obtained by plotting the molecular weight of five protein standards against their Kav (proportion of pores available to the molecule) for this column (black dots).

Figure 5

Reduced sensitivity to ABA or the ABA receptor-agonist CB in Nb high-order pyl mutants. (A) ABA-mediated inhibition of seedling establishment and early seedling growth is less in pyl1b pyl8a pyl8b pyl8c than in the WT. Seeds of the WT and mutant plants were germinated in MS medium lacking or supplemented with 1 μM ABA. Seedling establishment was scored after 9 days, and early seedling growth was scored after 15 days. The growth of the seedlings was quantified using IMAGE-J to obtain seedling projected area as a parameter for measuring growth. (B) CB-mediated inhibition of germination and seedling establishment is less in pyl1a pyl1b pyl8a pyl8b than in the WT. The asterisk indicates: * p ≤ 0.05 and ** p ≤ 0.01 (Student’s t-test or ANOVA followed by Dunnet’s test) when comparing data of mutant lines with non-transformed WT plants in the same assay conditions.

Figure 6

ABA-mediated inhibition of LR formation is reduced in pyl1b pyl8a pyl8b pyl8c relative to WT. (A) Inhibition of LR development and growth was scored in seedlings that were grown for 10 days in MS medium lacking or supplemented with 10 μM ABA. The number of lateral roots as well their length was scored using Image J. (B) PR growth was similar in the pyl1b pyl8a pyl8b pyl8c and WT plants. (C) Representative photographs of seedlings that were grown for 7 days in MS medium and then transferred to medium lacking or supplemented with 10 μM ABA. The pyl1b pyl8a pyl8b pyl8c and WT plants show differential growth of LRs in ABA medium. The asterisk indicates p ≤ 0.05 (Student’s t-test) when comparing data of mutant lines with non-transformed WT plants in the same assay conditions.

Figure 7

ABA-mediated inhibition of leaf growth is reduced in pyl1b pyl8a pyl8b pyl8c relative to WT. Leaf growth was scored in seedlings (n = 10) that were transferred from MS medium to medium lacking or supplemented with 10 μM ABA and grown for an additional 16 days. Total fresh weight (FW) of the mutant was measured and expressed as a percentage of FW relative to the WT in MS medium (100%, left panel) or in MS medium supplemented with ABA relative to the growth in MS medium lacking ABA (right panel). The asterisk indicates: * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001 (Student’s t-test or ANOVA followed by Dunnet’s test) when comparing data of mutant lines with non-transformed WT plants in the same assay conditions.