Overexpression of housekeeping gene FveIPT2 enhances anthocyanin and terpenoid accumulation in strawberry fruits with minimal impact on plant growth and development

Abstract

Anthocyanins and terpenoids are secondary metabolites with well-documented health benefits. Isopentenyl transferases (IPTs) are key enzymes in cytokinin (CK) biosynthesis. While ADP/ATP-type IPTs and their associated trans-zeatin (tZ)-CKs and iP-CKs are considered to play regulatory roles in growth and development, as well as stress acclimation in plants, tRNA-type IPTs and cis-zeatin CKs (cZ-CKs), which may serve housekeeping functions, remain less studied. In this study, the tRNA-type IPT gene FveIPT2 was overexpressed in woodland strawberries (Fragaria vesca). Overexpression had minimal impact on plant growth and CK levels but resulted in transgenic fruits exhibiting a significant increase in total phenolic, flavonoid, and anthocyanin contents, indicating enhanced fruit quality. Metabolite profiling revealed substantial increases in nine specific anthocyanins and 24 out of 47 detected terpenoids in the transgenic fruits. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis confirmed the upregulation of genes involved in anthocyanin and terpenoid biosynthesis and transport. These findings demonstrate that while tRNA-type IPTs may primarily play housekeeping roles, FveIPT2 overexpression can significantly enhance fruit quality by boosting terpenoid and anthocyanin accumulation, highlighting the unexpected potential of these genes to improve the nutritional value of edible fruits.

Figure 1

Overexpression of 35S::FveIPT2 in strawberry has minimal impact on plant growth, morphology, and cytokinin levels. No noticeable effects were observed in the 35S::FveIPT2 transgenic plants (40-day-old seedlings were shown in A and 180-day-old seedlings were shown in B). WT: wild type; FveIPT2-ox1 and FveIPT2-ox12 are 35S::FveIPT2 transgenic plants. Bar = 5 cm in (B). (C) FveIPT2 was expressed at high levels in 40-day-old transgenic seedlings. Error bars are SD (n = 3). P < 0.05. (D) A slight elevation in the concentrations of certain CKs was observed in 40-day-old transgenic seedlings. Error bars are SD (n = 3). P < 0.05.

Figure 2

Overexpression of 35S::FveIPT2 in strawberry enhanced fruit quality. Measurements of single fruit weight (A) and fruit shape index (B). (C) Fruit phenotype. Overexpression of FveIPT2 did not affect the levels of fructose (D), glucose (E) or sucrose (F). Overexpression of FveIPT2 elevated the levels of total phenolic (G), total flavonoid (H) and total anthocyanin (I). Error bars are SD (n = 3). P < 0.05.

Figure 3

The levels of amino acids and their derivatives, and flavonoids were significantly altered in the FveIPT2-ox12 transgenic fruits. (A) 666 metabolites were increased and 31 metabolites were reduced in transgenic fruits. Red-stage fruits (without seeds) from both WT and FveIPT2-ox12 plants were used for quasi-targeted metabolomics analysis. The criteria used to identify DEMs were as follows: VIP > 1.0, FC > 1.5 or FC < 0.667 and P-value < 0.05. (B) Volcano plot of DEMs between FveIPT2-ox12 and WT. The size of each dot reflects the VIP value. (C) The classification of DEMs revealed substantial alterations in amino acids and their derivatives, as well as flavonoids, in the fruits of FveIPT2-ox12 compared to those of WT. The numbers in parentheses indicate the counts of DEMs.

Figure 4

Overexpression of 35S::FveIPT2 in strawberry enhanced anthocyanin production. (A)Anthocyanin production was enhanced in transgenic fruits. Red-stage fruits (without seeds) from both WT and FveIPT2-ox12 plants were subjected to quasi-targeted metabolomics analysis. Each value in the heatmap represents the log₂-transformed original value of the anthocyanins. (B) The expression levels of anthocyanin-related genes were elevated in fruits from transgenic plants. Pre-turning stage fruits (without seeds) were used for RT-qPCR analysis, with FveCHC serving as the internal reference. Error bars are SD (n = 3). P < 0.05.

Figure 5

Overexpression of 35S::FveIPT2 in strawberry elevated terpenoid accumulation in fruits. (A) Terpenoid production was elevated in transgenic fruits. Red-stage fruits (without seeds) from both WT and FveIPT2-ox12 plants were subjected for quasi-targeted metabolomics analysis. Each value in the heatmap represents the log₂-transformed original value of terpenoids. (B) Linalool content was elevated, while α-pinene was decreased in FveIPT2-ox12 fruits. The volatile terpenoid contents in red fruits of both FveIPT2-ox12 and WT were detected by GC–MS. P < 0.05. (C) The expression levels of several genes associated with terpenoid production were significantly altered in transgenic fruits. Pre-turning stage fruits (without seeds) were used for RT-qPCR analysis, with FveCHC serving as the internal reference. Error bars are SD (n = 3). P < 0.05.

Figure 6

Altered expression levels of CK-regulated genes in 35S::FveIPT2 transgenic fruits. CK-responsive genes were downregulated in transgenic fruits. Pre-turning stage fruits (without seeds) were used for RT-qPCR analysis, with FveCHC serving as the internal reference. Error bars are SD (n = 3). P < 0.05.