Polyamines Regulate Strawberry Fruit Ripening by Abscisic Acid, Auxin, and Ethylene

Abstract

Polyamines (PAs) participate in many plant growth and developmental processes, including fruit ripening. However, it is not clear whether PAs play a role in the ripening of strawberry (Fragaria ananassa), a model nonclimacteric plant. Here, we found that the content of the PA spermine (Spm) increased more sharply after the onset of fruit coloration than did that of the PAs putrescine (Put) or spermidine (Spd). Spm dominance in ripe fruit resulted from abundant transcripts of a strawberry S-adenosyl-l-Met decarboxylase gene (FaSAMDC), which encodes an enzyme that generates a residue needed for PA biosynthesis. Exogenous Spm and Spd promoted fruit coloration, while exogenous Put and a SAMDC inhibitor inhibited coloration. Based on transcriptome data, up- and down-regulation of FaSAMDC expression promoted and inhibited ripening, respectively, which coincided with changes in several physiological parameters and their corresponding gene transcripts, including firmness, anthocyanin content, sugar content, polyamine content, auxin (indole-3-acetic acid [IAA]) content, abscisic acid (ABA) content, and ethylene emission. Using isothermal titration calorimetry, we found that FaSAMDC also had a high enzymatic activity with a K_d of 1.7 × 10^-3 m In conclusion, PAs, especially Spm, regulate strawberry fruit ripening in an ABA-dominated, IAA-participating, and ethylene-coordinated manner, and FaSAMDC plays an important role in ripening.

Figure 1.

Polyamine contents in seven developmental stages of strawberry fruit. Polyamine contents examined using HPLC. Three fruits were used for detection in each stage. Error bars represent se (n = 3).

Figure 2.

Effects of polyamines and an inhibitor on strawberry fruit coloration. Twenty degreening strawberry fruits still attached to the plants were used for each independent treatment and immersed in 100 µm Put, Spd, Spm, or MGBG (n = 20, one replication). Water was used as the control. Three fruits representative of the Put-, Spm-, Spd-, and MGBG-treated phenotypes were used for detection of the physiological parameters (n = 3, three replications). A, Fruit phenotypes before (DG) and after treatment. B, The number of fruits per phenotype within each treatment. C, Anthocyanin contents. D, Polyamine contents. E, Ethylene emission rates. F, ABA contents. G, IAA contents. Error bars represent se (n = 3).The asterisk in same-colored columns indicates a statistically significant difference compared to the control (P < 0.05) after an ANOVA followed by Duncan’s multiple range tests.

Figure 3.

DEGs during the onset of strawberry fruit ripening as determined by RNA-seq. Fruit cDNA libraries from four developmental stages, including LG, Wt, IR, and PR, were used for RNA-seq. A, The number of upregulated and downregulated DEGs between library pairs. B, Heat map and cluster analysis of the DEGs encoding proteins in plant hormone signaling pathways. The data for gene expression levels were normalized to a z-score with the formula log₁₀ (FPKM+1) by color key and density plot. Green represents low expression and red represents high expression during ripening. IAA, auxin-responsive protein/transcription factor; LUX, auxin transporter 2; JAZ, jasmonate ZIM (zinc-finger inflorescence meristem) domain; PP2C37/ABI37, 2C-type protein phosphatase 37/ ABA-insensitive 37; EBF1, EIN3 (ethylene insensitive 3) binding F-box 1; GID1, gibberellin receptor 1; ACO1, 1-aminocyclopropane-1-carboxylic acid oxidase 1; SnRK2, Ser/Thr-protein kinase 2.

Figure 4.

Transcripts of FaSAMDC during strawberry fruit ripening as determined by RNA-seq and qPCR expression analysis. A, Eight SAMDC gene-like contigs were found in the transcriptomic data from LG, Wt, IR, and PR fruits. B, The mRNA expression levels of FaSAMDC in six fruit stages, including LG, DG, Wt, IR, PR, and FR. Actin mRNA was used as an internal control. Columns with different letters (a–f) indicate statistically significant differences (P < 0.05) after an ANOVA followed by Duncan’s multiple range tests. Error bars represent se (n = 3).

Figure 5.

Silencing and overexpression of FaSAMDC in developing strawberry fruit. Twenty DG fruit still attached to the plant were used for inoculations. A to C, Eight days after inoculation, FaSAMDC-VIGS (RNAi) fruit (A) showed chimeric phenotypes compared to the control (B), while the FaSAMDC-OE fruits (C) appeared dark red. D, FaSAMDC transcripts in RNAi and OE fruit compared to the control. E, TRV vector detection in VIGS fruit. Actin mRNA was used as an internal control. The asterisk in the columns indicates statistically significant differences (P < 0.05) compared to the control after an ANOVA followed by Duncan’s multiple range tests. Error bars represent se (n = 3).

Figure 6.

Manipulation of FaSAMDC expression affects several physiological parameters and relevant gene transcripts. The FaSAMDC transgenic fruits, in which the target gene was down (RNAi)- and up (OE)-regulated by more than 80% compared with the mixed control fruits, were used for analysis. A, Polyamine contents including Put, Spd, and Spm. B, Ethylene emissions. C, ABA and IAA contents. D, Fruit firmness. E, Anthocyanin contents. F, Soluble sugar contents. G, Ripening-related gene expression levels, including firmness (polygalacturonase [PG1] and pectate lyase [PL1]), anthocyanin (chalcone synthase [CHS] and dihydroflavonol 4-reductase [DFR]), sugar (Suc transporter 1 [SUT1] and Suc synthase [SS]), polyamines (Arg decarboxylase [ADC], Orn decarboxylase [ODC], Spd synthase [SPDS], and Spm synthase [SPMS]), IAA (auxin-responsive protein 2 and factor 11 [IAA2 and ARF11], and auxin transporter 2 [LUX2]), ABA (protein phosphatase 2C 37 [ABI37], Ser/Thr-protein kinase SRK2 [SnRK2], and 9-cis-epoxycarotenoid dioxygenase [NCED1]), and ethylene (1-aminocyclopropanecarboxylate synthase 1 [ACO1] and EIN3-binding F-box protein [EBF1]) genes. Actin mRNA was used as an internal control. The asterisk in the same color or gene columns indicates statistically significant differences (P < 0.05) compared to the control after an ANOVA followed by Duncan’s multiple range tests. Error bars represent se (n = 3).

Figure 7.

Purification, identification, and enzymatic activity of FaSAMDC protein. A, Purification of the 66-kD recombinant FaSAMDC protein. B, Immunoblot identification of the recombinant FaSAMDC. C, Measurement of the affinity between SAM and the purified FaSAMDC protein using isothermal titration calorimetry. A typical and specific saturation curve with stoichiometry (N) of 1:1 was obtained, suggesting that one SAM molecule could bind per purified protein molecule with a dissociation constant of 1.7 × 10⁻³ m.

Figure 8.

A model for PA regulation of strawberry fruit ripening through ABA, IAA, and ethylene. In developing strawberry fruit, the biosynthesis of polyamines (Put, Spd, and Spm) depends on ODC, ADC, SAMDS, SPDS, and SPDM, of which SAMDC is a rate-limiting step for Spd and Spm synthesis. The up-regulation of SAMDC expression positively regulates the expression of ODC, ADC, SPDS, and SPDM, which promotes Spd and, especially, Spm accumulation while inhibiting Put accumulation. A high ratio of (Spd + Spm)/Put accelerates ABA synthesis and signaling by promoting the expression of NCED1 (key to ABA synthesis) and SnRK2 (a positive regulator of ABA signaling) and inhibiting the expression of ABI37 (a negative regulator of ABA signaling).The high (Spd + Spm)/Put ratio also differently regulates IAA transport and signaling by inhibiting the expression of AUX2 (key to ABA transport) and IAA2 (a negative regulator of IAA signaling) and promoting the expression of ARF11 (a positive regulator of ABA signaling). However, the high ratio inhibits ethylene synthesis and signaling by inhibiting the expression of ACO1 (key to ethylene synthesis) gene and promoting the expression of EBF1 (a negative regulator of ethylene signaling) gene. Thus, PAs regulate strawberry fruit ripening in an ABA-dominated, IAA-participating, and ethylene-coordinated manner to promote ripening-related gene expression levels, including those for firmness (PG1 and PL1), anthocyanin content (CHS and DFR), and sugar content (SUT1 and SS). The graphic symbols (arrow, t-bar, and arrow with bar) represent promote, inhibit, and no cooperation. Red arrows indicate promotion.