Archetypal Roles of an Abscisic Acid Receptor in Drought and Sugar Responses in Liverworts

Abstract

Abscisic acid (ABA) controls seed dormancy and stomatal closure through binding to the intracellular receptor Pyrabactin resistance1 (Pyr1)/Pyr1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) in angiosperms. Genes encoding PYR/PYL/RCAR are thought to have arisen in the ancestor of embryophytes, but the roles of the genes in nonvascular plants have not been determined. In the liverwort Marchantia polymorpha, ABA reduces growth and enhances desiccation tolerance through increasing accumulation of intracellular sugars and various transcripts such as those of Late Embryogenesis Abundant (LEA)-like genes. In this study, we analyzed a gene designated MpPYL1, which is closely related to PYR/PYL/RCAR of angiosperms, in transgenic liverworts. Transgenic lines overexpressing MpPYL1-GFP showed ABA-hypersensitive growth with enhanced desiccation tolerance, whereas Mppyl1 generated by CRISPR-Cas9-mediated genome editing showed ABA-insensitive growth with reduced desiccation tolerance. Transcriptome analysis indicated that MpPYL1 is a major regulator of abiotic stress-associated genes, including all 35 ABA-induced LEA-like genes. Furthermore, these transgenic plants showed altered responses to extracellular Suc, suggesting that ABA and PYR/PYL/RCAR function in sugar responses. The results presented here reveal an important role of PYR/PYL/RCAR in the ABA response, which was likely acquired in the common ancestor of land plants. The results also indicate the archetypal role of ABA and its receptor in sugar response and accumulation processes for vegetative desiccation tolerance in bryophytes.

Figure 1.

PYR/PYL/RCAR-Like Genes in Marchantia polymorpha. A, Phylogenetic analysis of PYR/PYL/RCAR-related proteins in Arabidopsis, Amborella trichopoda, Picea abies, Selaginella moellendorffii, Physcomitrella patens, and M. polymorpha. Amino acid sequences were aligned with MUSCLE. Phylogenetic relationship among PYR/PYL/RCAR-like proteins was inferred using the neighbor-joining method. Numbers on the branches indicate bootstrap values (100 replicates). The bar represents the number of amino acid changes per branch length. Phylogenetic analyses were conducted in MEGA7. B, Effect of overexpression of MpPYL1 on ABA-induced gene expression. The cDNA of MpPYL1 fused with the rice actin promoter was introduced into the P. patens protonemata cells by particle bombardment. The ABA-inducible Em promoter fused with the GUS reporter gene was used as a reporter construct. The tissues were incubated with or without ABA (10 µM) and used for fluorometric GUS assays. GUS activity was normalized by expression of the cointroduced luciferase (LUC) reporter gene. Results of PYL6 of Arabidopsis (AtPYL6, At2g40330) are shown for comparison. *P < 0.05 and ***P < 0.001 by the t test.

Figure 2.

MpPYL1-Enhanced Gene Expression Is Mediated by ABRE. A, Transient assays of Physcomitrella patens protonemata cells using the GUS reporter gene fused to the nonmutated Em promoter and the promoters with mutations in ABRE (mABRE), RY (mRY), or both (mABRE/mRY). B, Enhanced expression by MpPYL1 of GUS fused with the 4xABRE promoter (_pro4xABRE-GUS). The GUS construct without the promoter (GUS) was used as a control. GUS expression was normalized by the cointroduced LUC gene. Error bars indicate se (n = 3). *P < 0.05 by the t test.

Figure 3.

Enhanced ABA Sensitivity in MpPYL1-GFP Overexpression Lines of Transgenic Marchantia polymorpha. A, Two transgenic lines, MpPYL1ox8 and MpPYL1ox9, were subjected to immunoblot analysis using an anti-GFP antibody. Molecular size markers are shown in kilodalton (kDa). B and C, Growth of TAK1 (the wild type) and the transgenic lines on a medium containing 0 to 5 µM ABA are shown by area (B) and appearance (C) of the thalli. Error bars indicate se (n = 3). *P < 0.05 compared with TAK1 of the same treatment by the t test. Long-term effect of ABA on growth of the thalli is shown in Supplemental Fig. S4.

Figure 4.

Generation and Growth Analysis of MpPYL1 Genome-Edited Lines. A, Nucleotide sequences of the MpPYL1 genome region of TAK1 (the wild type) and genome-edited lines Mppyl1^ge1b, Mppyl1^ge1c, Mppyl1^ge2b, and Mppyl1^ge2c. A hyphen represents a deletion of a nucleotide in the genome-edited lines. B, Fresh weight of gemmalings cultured on medium containing 10 µM ABA for two weeks. Error bars indicate the se (n = 3). *P < 0.05 compared with the minus ABA control by the t test. C, Appearance of the representative gemmalings shown in (B). Effects of higher concentration of ABA on growth are shown in Supplemental Fig. S5.

Figure 5.

Transcriptome Analysis of MpPYL1-Dependent Genes. A, Venn diagrams showing the number of MpPYL1-regulated genes revealed by RNA-seq analysis. The upper diagram shows relationships between 567 genes induced by ABA in TAK1 (the wild type), 397 genes for which expression in ABA-treated MpPYL1ox8 was more than 2-fold of that in ABA-treated TAK1 and 1013 genes for which expression in ABA-treated Mppyl1^ge2b was less than 2-fold of that in ABA-treated TAK1. The lower diagram shows relationships between 378 genes repressed by ABA in TAK1, 182 genes for which expression in ABA-treated MpPYL1ox8 was less than 2-fold of that in ABA-treated TAK1, and 423 genes for which expression in ABA-treated Mppyl1^ge2b was more than 2-fold of that in ABA-treated TAK1. B, A heatmap shows hierarchical clustering of transcripts. Gene expression values are represented as relative to the mean of all samples; blue color represents lower expression, and red color represents higher expression.

Figure 6.

ABA-Induced Expression of LEA-Like Genes in TAK1, MpPYL1ox8, and Mppyl1^ge2b. A, Venn diagram showing expression of LEA-like genes in TAK1 (the wild type) and expression of 38 genes in MpPYL1ox8 and Mppyl1^ge2b. The diagram shows the relationship between 35 LEA-like genes for which expression was induced by ABA in TAK1, 33 genes for which expression was increased in MpPYL1ox8, and 38 genes for which expression was decreased in Mppyl1^ge2b. B, RT-PCR analysis of RNA isolated from gemmalings treated with 1 µM ABA for 6 h. Primers for MpLEAL1 (Mapoly0112s0030), MpLEAL2 (Mapoly0119s0008), MpLEAL3 (Mapoly0035s0082), MpLEAL4 (Mapoly0016s0148), MpLEAL5 (Mapoly0087s0015), MpLEAL6 (Mapoly0027s0114), and MpLEAL7 (Mapoly0025s0042) were used for amplification. Results for MpEF1 (Mapoly0024s0116) unchanged by ABA and ethidium bromide-stained rRNA bands are shown for comparison.

Figure 7.

Sensitivity of Growth to Suc in Gemmalings of TAK1, MpPYL1ox8, and Mppyl1^ge2b lines. A, Growth appearance on media containing 0, 0.1, and 0.2 m Suc after 2 weeks of culture. B, Fresh weight measurement of plants shown in (A). Error bars indicate se (n = 3). *P < 0.05 by the t test.

Figure 8.

Effects of ABA and Suc on Gene Expression and Sugar Accumulation. A, ABA- and Suc-induced gene expression in gemmalings of TAK1, MpPYL1ox8, and Mppyl1^ge2b. Gemmalings were cultured for 6 h in a medium with or without 0.1 m Suc, and 1 µM ABA were used for RNA extraction. The extracted RNA was used for RT-PCR analysis using primers of LEA-like genes, MpLEAL1 (Mapoly0112s0030), MpLEAL2 (Mapoly0119s0008), and MpLEAL3 (Mapoly0035s0082). Results for MpEF1 (Mapoly0024s0116) and ethidium bromide-stained rRNA bands are shown for comparison. B and C, ABA-induced sugar accumulation is shown in gemmalings of TAK1, MpPYL1ox8, and Mppyl1^ge2b. Gemmalings were cultured for one day with or without 1 µM ABA in a half-strength B5 medium containing no sugar (B) or 0.1 m Suc (C). The extracted sugars were used for quantitation by anthrone assays with Glc as a standard. Error bars indicate se (n = 3). *P < 0.05 by the t test.

Figure 9.

Desiccation Tolerance of Gemmalings of TAK1, MpPYL1ox8, and Mppyl1^ge2b. Gemmalings cultured with or without 10 µM ABA (+ABA) and 0.1 m Suc (+Suc) were desiccated in a container with silica gel for two days in the dark. The rehydrated gemmalings were transferred onto agar medium and cultured for two weeks.