Overexpression of IbFAD8 Enhances the Low-Temperature Storage Ability and Alpha-Linolenic Acid Content of Sweetpotato Tuberous Roots

Abstract

Sweetpotato is an emerging food crop that ensures food and nutrition security in the face of climate change. Alpha-linoleic acid (ALA) is one of the key factors affecting plant stress tolerance and is also an essential nutrient in humans. In plants, fatty acid desaturase 8 (FAD8) synthesizes ALA from linoleic acid (LA). Previously, we identified the cold-induced IbFAD8 gene from RNA-seq of sweetpotato tuberous roots stored at low-temperature. In this study, we investigated the effect of IbFAD8 on the low-temperature storage ability and ALA content of the tuberous roots of sweetpotato. Transgenic sweetpotato plants overexpressing IbFAD8 (TF plants) exhibited increased cold and drought stress tolerance and enhanced heat stress susceptibility compared with non-transgenic (NT) plants. The ALA content of the tuberous roots of TF plants (0.19 g/100 g DW) was ca. 3.8-fold higher than that of NT plants (0.05 g/100 g DW), resulting in 8-9-fold increase in the ALA/LA ratio in TF plants. Furthermore, tuberous roots of TF plants showed better low-temperature storage ability compared with NT plants. These results indicate that IbFAD8 is a valuable candidate gene for increasing the ALA content, environmental stress tolerance, and low-temperature storage ability of sweetpotato tuberous roots via molecular breeding.

Keywords: IbFAD8; abiotic stress; alpha-linolenic acid; low temperature; sweetpotato; tuberous roots.

FIGURE 1

Amino acid sequence analysis of IbFAD8. (A) Alignment of the predicted amino acid sequences of FAD8 and FAD7 of various plant species. Ib, Ipomoea batatas; At, Arabidopsis thaliana, Os, Oryza sativa. (B) Phylogenetic analysis of the deduced amino acid sequences of plastidial FAD enzymes (FAD7 and FAD8) of various plant species. GenBank accession numbers of FAD sequences are as follows: NtFAD7 (BAC01274.1), OeFAD7-1 (AFX68461.1), SiFAD7 (NP_001306619.1), AtFAD7 (NP_187727.1), AtFAD8 (NP_196177.1), StFAD (NP_001274883.1), LeFAD7 (NP_001234592.1), GmFAD8-1 (NP_001238609.1), VuFAD8 (ABY60737.1), VuFAD7 (ABY60738.1), ZmFAD (BAA22441.1), ZmFAD8 (NP_001266540.1), OsFAD7 (BAE79783.1), OsFAD8 (AAW32557.1). (C) Subcellular localization of IbFAD8. The IbFAD8-GFP fusion was transiently expressed in Nicotiana benthamiana leaves. Confocal microscopy was used to visualize the GFP signal (green) and chlorophyll a autofluorescence (red). Merged images were used to confirm the subcellular localization of IbFAD8.

FIGURE 2

Analysis of the expression levels of IbFAD8 in sweetpotato. (A) Expression levels of IbFAD8 in different tissues of sweetpotato plants. The Ubiquitin gene was used as an internal control for the normalization of IbFAD8 expression levels. L, leaves; S, stems; FR, fibrous roots; PR, pencil roots; TR, tuberous roots. (B) Position-dependent leaf development in sweetpotato plants. Images show representative leaves at each position from the shoot apical meristem. (C) Transcript levels of IbFAD8 in leaves at different positions on the stem. (D) Expression levels of IbFAD8 in sweetpotato tuberous roots at different developmental stages. FR, fibrous roots; PR, pencil roots; TR1, tuberous roots (diameter: 1–1.5 cm); TR1.5, tuberous roots (diameter: 1.5–2 cm); TR2, tuberous roots (diameter > 2 cm). (E) Transcript levels of IbFAD8 in sweetpotato leaves and (F) tuberous roots in response to various abiotic stresses. The third and fourth leaves from the top of sweetpotato plants were treated with cold (4°C) and heat (42°C) stresses, and IbFAD8 expression was analyzed by qRT-PCR. To analyze the effects of drought stress, the detached third leaf was placed in a 1.5-mL microfuge tube containing 30% polyethylene glycol (PEG 6000). Tuberous roots were stored at 4°C for 6 weeks to investigate IbFAD8 expression levels. All the experiments were conducted three times with 3 individual tissues for each replicate. h, hours; w, weeks. Different lowercase letters indicate significant differences (p < 0.05; one-way ANOVA, followed by Tukey’s HSD post hoc test).

FIGURE 3

Generation of transgenic sweetpotato plants overexpressing IbFAD8. (A) Schematic diagram of the vector used to generate transgenic sweetpotato lines. The IbFAD8 gene was expressed under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter. (B) Gel image showing the results of genomic DNA (gDNA) PCR. PC, positive control; NT, non-transgenic plants. (C) Expression levels of IbFAD8 in the leaves of transgenic lines. Lines #5 and #6 with high transcript levels (referred to as TF5 and TF6, respectively) were selected for further characterization.

FIGURE 4

Evaluation of the abiotic stress tolerance of TF plants. (A) Phenotypic changes in TF plants exposed to cold (4°C) stress for 70 h, followed by recovery at 25°C for 60 h. (B,C) Fv/Fm values (B) and malondialdehyde (MDA) contents (C) of sweetpotato leaves after cold stress treatment and recovery. (D) Phenotypic analysis of TF plants treated with heat stress (42°C) for 60 h, followed by recovery for 72 h. (E,F) Fv/Fm values (E) and MDA contents (F) of sweetpotato leaves after heat stress treatment and recovery. (G) Phenotypes of TF plants treated with drought stress for 12 day, followed by rehydration for 6 day. (H,I) Chlorophyll contents (SPAD units) (H) and MDA contents (I) of sweetpotato leaves during drought stress treatment and recovery. Asterisks indicate significant differences between TF and NT plants (*p < 0.05, **p < 0.01).

FIGURE 5

Evaluation of the low-temperature storage ability of the tuberous roots of sweetpotato plants. (A) Phenotypes of tuberous roots stored at normal temperature (13°C) and low temperature (4°C). Whole tuberous roots (upper panel) and root sections (lower panel) were photographed. (B–D) Quantification of MDA level (B), hydrogen peroxide (H₂O₂) level (C), and specific peroxidase (POD) activity (D) in tuberous roots after storage. Asterisks indicate significant differences between NT and TF plants (*p < 0.05, **p < 0.01; one-way ANOVA, followed by Tukey’s HSD post hoc test).