FaPOD27 functions in the metabolism of polyphenols in strawberry fruit (Fragaria sp.)

Abstract

The strawberry (Fragaria × ananassa) is one of the most preferred fresh fruit worldwide, accumulates numerous flavonoids but has limited shelf life due to excessive tissue softening caused by cell wall degradation. Since lignin is one of the polymers that strengthen plant cell walls and might contribute to some extent to fruit firmness monolignol biosynthesis was studied in strawberry fruit. Cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), and a peroxidase (POD27) gene were strongly expressed in red, ripe fruit whereas a second POD gene was primarily expressed in green, immature fruit. Moreover, FaPOD27 transcripts were strongly and constitutively induced in fruits exposed to Agrobacterium infection. Gene expression levels and enzymatic activities of FaCCR and FaCAD were efficiently suppressed through RNAi in FaCCR- and FaCAD-silenced strawberries. Besides, significantly elevated FaPOD transcript levels were detected after agroinfiltration of pBI-FaPOD constructs in fruits. At the same time, levels of G-monomers were considerably reduced in FaCCR-silenced fruits whereas the proportion of both G- and S-monomers decisively decreased in FaCAD-silenced and pBI-FaPOD fruits. Development, firmness, and lignin level of the treated fruits were similar to pBI-intron control fruits, presumably attributed to increased expression levels of FaPOD27 upon agroinfiltration. Additionally, enhanced firmness, accompanied with elevated lignin levels, was revealed in chalcone synthase-deficient fruits (CHS(-)), independent of down- or up-regulation of individual and combined FaCCR. FaCAD, and FaPOD genes by agroinfiltration, when compared to CHS(-)/pBI-intron control fruits. These approaches provide further insight into the genetic control of flavonoid and lignin synthesis in strawberries. The results suggest that FaPOD27 is a key gene for lignin biosynthesis in strawberry fruit and thus to improving the firmness of strawberries.

Keywords: fruit firmness; lignification; monolignol genes; peroxidase; strawberry.

Figure 1

Flavonoid and monolignol biosynthetic pathways. PAL, phenylalanine ammonia lyase; 4CL, 4-coumaroyl-CoA ligase; CHS, chalcone synthase; HCT, hydroxycinnamoyl transferase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase; POD, peroxidase. Arrow with dashed lines indicate compounds are up- () or down-regulated () when fruits were agroinfiltrated pBI-intron (control constructs), down- or up-regulation of individual genes (FaCCR. FaCAD, or FaPOD) (Fig. S12). The green shade indicating p-coumaroyl-CoA is the common substrate of CHS, HCT, and CCR. Red CCR. CAD, and POD indicate down- and up-regulated genes.

Figure 2

Relative expression profiles of monolignol biosynthesis genes in vegetative tissues, flowers, and fruit developmental stages of F. × ananassa cv. Elsanta. Total RNA was isolated from (A) fruit developmental stages at small green (SG), green white (GW), white (W), turning (T), and red (R) after pollination. (B) Expression levels of vegetative tissues (leaf; stem; runner; root), flower (F) and fruit developmental stages (SG, GW, W, T, and R) were monitored by qRT-PCR. FaCCR. FaCAD. FaPOD and FaPOD27 (Ring et al., 2013) were target genes. The white fruit was used as the reference with one for each graph. Values are mean ± SE of 5–6 replicates from two sets of cDNAs and are shown as relative changes.

Figure 3

Relative expression profiles of phenylpropanoid biosynthesis genes of F. × ananassa cv. Elsanta in response to Agrobacterium. Expression levels in control (gray column) and agroinfiltrated fruits (black column) were monitored by qRT-PCR at different time points (1, 3, 6, 12, 24, 48, and 96 h). FaPAL. FaCHS, FaCCR. FaCAD. FaPOD, and FaPOD27 were target genes. The control fruit (1 h) was used as the reference with one for each graph. Values are mean ± SE of 2–3 replicates from one fruit and are shown as relative changes.

Figure 4

Effect of wounding and pathogen infection on fruit firmness of F. × ananassa cv. Elsanta. (A) WT (Wild type; n = 10); fruits infiltrated with MMA medium (wounded; n = 12) and Agrobacterium (pathogen; n = 10). (B) The Wilcoxon-Mann-Whitney U-test was used for a non-parametric comparison of two groups from WT and wounded or pathogen-infected fruits. Values showing statistically significant increased levels (P < 1.00E-02) are marked by a yellow background.

Figure 5

Schematic diagram of constructs (A), and phenotype (I) and lignin staining (II) of wild-type and infiltrated fruits (B). (A) All constructs are in the binary vector pBI121. CaMV 35S, the 35S promoter of cauliflower mosaic virus; intron, the second intron of strawberry FaQR gene (Raab et al., 2006); NOS, terminator of the nopaline synthase gene. A control construct (pBI-intron) contained a GUS gene interrupted by an intron; an ihpRNA-cassette construct contained an intron flanked by partial coding sequences (300 bp) of target genes (FaCCR. FaCAD, or FaPOD) in sense and antisense orientations; an overexpression-cassette comprised the full-length coding sequence of target genes (FaCCR. FaCAD, or FaPOD). All constructs were used for agroinfiltration of fruits. (B) All photographs were taken 14 days after infiltration. WT (Wild type; a, c) was used as a non-infiltrated fruit. Infiltrated fruits are those injected with Agrobacterium suspensions harboring different constructs, as described in (A). (I) Phenotypes of WT (a, c) and infiltrated fruits (b, d). (II) Cross-sections of WT (a, c; n = 2) and infiltrated fruits (b, d; n = 2) before and after Wiesner staining. The arrow indicates native lignins in the fruit infiltrated with Agrobacterium suspensions harboring different constructs, as described in (A).

Figure 6

Fruit firmness (A,B) and lignin content (C,D) of individual FaCCR-. FaCAD-. FaPOD-downregulation (A,C) and -upregulation (B,D) in F. × ananassa cv. Elsanta. (A) Firmness of WT (wild type; n = 26), control fruits (pBI-intron; n = 41), and down-regulated fruits (pBI-FaCCRi; n = 32, pBI-FaCADi; n = 30, pBI-FaPODi; n = 35); (B) Firmness of WT (n = 30), pBI-intron (n = 41), and up-regulated fruits (pBI-FaCCR; n = 39, pBI-FaCAD; n = 35, pBI-FaPOD; n = 43); (C) Lignin content of WT (n = 14), pBI-intron (n = 19), pBI-FaCCRi (n = 19), pBI-FaCADi (n = 19), and pBI-FaPODi (n = 19); (D) Lignin content of each group (n = 10). The statistical analysis methodology used was the Wilcoxon-Mann-Whitney U-test for a non-parametric comparison of two groups. One asterisk (^*) marked in the box indicates statistically significant increased levels (P < 0.01) in comparison with WT and another group.

Figure 7

Relative expression profiles of down- (A) or up-regulated (B) monolignol genes in F. × ananassa cv. Elsanta. Total RNA was isolated from a single untreated fruit (wild type; WT), control fruits (pBI-intron), down-regulated fruits (pBI-FaCCRi, pBI-FaCADi, and pBI-FaPODi) (A), and up-regulated fruits (pBI-FaCCR, pBI-FaCAD, and pBI-FaPOD) (B). Expression levels of all samples were monitored by qRT-PCR with specific primers for target genes (FaCCR. FaCAD, and FaPOD) and an interspacer gene. The latter was used as an internal control for normalization. For each box-plot graph, one of the WT group was used as the reference (set to one) and each group contained five biological replicates. The Wilcoxon-Mann-Whitney U-test was used for a non-parametric comparison of two groups from pBI-intron and fruits infiltrated with different constructs. Values indicate statistically decreased and increased levels (P < 0.05) and are shown in the box.

Figure 8

CCR (A,B) and CAD (C,D) specific activity in wild-type and agroinfiltrated fruits. (A) Coniferaldehyde (P; products) formed in the reactions was quantified at 340 nm. (B) Untreated fruits (WT), control fruits (pBI-intron), pBI-FaCCR, and pBI-FaCCRi; each group n = 7. (C) Coniferyl alcohol formed in the reactions (P = coniferyl alcohol; S = coniferaldehyde) was quantified at 260 nm. (D) WT, pBI-intron, pBI-FaCAD, and pBI-FaCADi fruits; each group n = 6. The Wilcoxon-Mann-Whitney U-test was used for a non-parametric comparison of two groups. One asterisk (^*) or two asterisks (^**) in the box mark statistically significant decreased levels (P < 0.02) in comparison with WT and another group (indicated by ^*), or in comparison with either WT or pBI-intron and another group (indicated by ^**).

Figure 9

Comparison of non-infiltrated and infiltrated Calypso (CHS⁻) fruits. All pictures were taken 14 days after infiltration. Phenotypes of non-infiltrated (A–D) and infiltrated fruits (E–H) are compared. Non-infiltrated fruits of Calypso with CHS genes (CHS⁺; A,B) and Calypso with impaired CHS genes (CHS⁻; C,D) are shown. Infiltrated fruits (E–H) represent Calypso fruit (CHS⁻) injected with Agrobacterium suspensions harboring either pBI-intron, FaCCR-. FaCAD-. FaPOD-ihpRNA, FaCCR-. FaCAD-, FaPOD-overexpression constructs, or combined pBI-Si3 and pBI-O3 constructs. All agroinfiltrated fruits showed a similar phenotype (E–H).

Figure 10

Fruit firmness (A,B) and lignin content (C,D) of FaCCR-. FaCAD-. FaPOD-downregulation and -upregulation as well as combinations in F. × ananassa cv. Calypso (CHS⁻). (A) Firmness of Calypso (CHS⁻; n = 15), control fruits (CHS⁻/pBI-intron; n = 17), down-regulated fruits (CHS⁻/pBI-FaCCRi; n = 14, CHS⁻/pBI-FaCADi; n = 14, CHS⁻/pBI-FaPODi; n = 17), and combined three genes (CHS⁻/pBI-Si3; n = 15). (B) Firmness of Calypso (CHS⁻, n = 15), control fruits (CHS⁻/pBI-intron; n = 17), up-regulated fruits (CHS⁻/pBI-FaCCR; n = 14, CHS⁻/pBI-FaCAD; n = 17, CHS⁻/pBI-FaPOD; n = 15), and combined three genes (CHS⁻/pBI-O3; n = 18). (C,D) Lignin content of each group (n = 5). The Wilcoxon-Mann-Whitney U-test was used for non-parametric analysis of two groups. One asterisk (^*) or a plus (+) in the box marks statistically significant increased levels (P < 0.05) in comparison with CHS⁻ and another group (indicated by ^*), or in comparison with CHS⁻/pBI-intron and another group (indicated by +).