Overexpression of the Golden SNP-Carrying Orange Gene Enhances Carotenoid Accumulation and Heat Stress Tolerance in Sweetpotato Plants

Carotenoids function as photosynthetic accessory pigments, antioxidants, and vitamin A precursors. We recently showed that transgenic sweetpotato calli overexpressing the mutant sweetpotato (Ipomoea batatas [L.] Lam) Orange gene (IbOr-R96H), which carries a single nucleotide polymorphism responsible for Arg to His substitution at amino acid position 96, exhibited dramatically higher carotenoid content and abiotic stress tolerance than calli overexpressing the wild-type IbOr gene (IbOr-WT). In this study, we generated transgenic sweetpotato plants overexpressing IbOr-R96H under the control of the cauliflower mosaic virus (CaMV) 35S promoter via Agrobacterium-mediated transformation. The total carotenoid contents of IbOr-R96H storage roots (light-orange flesh) and IbOr-WT storage roots (light-yellow flesh) were 5.4–19.6 and 3.2-fold higher, respectively, than those of non-transgenic (NT) storage roots (white flesh). The β-carotene content of IbOr-R96H storage roots was up to 186.2-fold higher than that of NT storage roots. In addition, IbOr-R96H plants showed greater tolerance to heat stress (47 °C) than NT and IbOr-WT plants, possibly because of higher DPPH radical scavenging activity and ABA contents. These results indicate that IbOr-R96H is a promising strategy for developing new sweetpotato cultivars with improved carotenoid contents and heat stress tolerance.


Introduction
Given their functional importance, carotenoids have been investigated since the beginning of the 19th century, and over 1100 carotenoid compounds have been identified to date [1].Tetraterpenoids are lipid-soluble carotenoid pigments produced by all photosynthetic organisms, including plants, algae, bacteria, and fungi.In plants, carotenoids are generally categorized into two classes, xanthophylls, and carotenes.These carotenoids are red, orange, and yellow-colored pigments, which are responsible for the coloration of fruits and flowers.These carotenoids also play a vital role in protecting the photosynthetic apparatus from photo-oxidation, which can trigger the formation of reactive oxygen species (ROS) [2,3].In addition to their functions in photosynthetic organisms, carotenoids also serve as precursors of pro-vitamin A, and exhibit antioxidant properties and the abil-ity to relieve chronic diseases in humans and animals [4,5].In developing countries, vitamin A deficiency affects approximately 130 million preschool-aged children and 7 million pregnant women and is responsible for the death of 0.6 million children aged under 5 years [6].To alleviate global health issues caused by the lack of micronutrients, several studies have been carried out to investigate the biofortification of plant-derived foods with carotenoid precursors of pro-vitamin A (mainly β-carotene); for example, Golden Rice, which contains a high level of carotenoids [7,8].These studies have led to the development of a number of biofortified crops using transgenic approaches by exploiting genes involved in the carotenogenesis pathway [9,10].
Sweetpotato (Ipomoea batatas [L.] Lam) is the seventh most important food crop in the world and a rich source of diverse nutrients [11].It is believed that sweetpotato, especially orange-fleshed cultivars, could combat vitamin A deficiency because of the high level of β-carotene (approximately 90% of all carotenoids) [12].Sweetpotato has a high nutritional value and is rich in low molecular weight antioxidants (LMWAs) such as carotenoids, ascorbate, tocopherols, and polyphenols [13,14].Sweetpotato is also considered a valuable source of unique natural products, including some that can be used for the development of medicines effective against various diseases and for the production of industrial products [15,16].To develop new sweetpotato cultivars with high LMWA content, genetic engineering of the biosynthetic and metabolic genes of LMWAs is currently in progress via molecular breeding.Metabolic engineering of carotenoid biosynthesis genes has been investigated to increase the accumulation of different types of carotenoids.Transgenic sweetpotato plants with enhanced carotenoid content also exhibit environmental stress tolerance [13,14].
The Orange (Or) gene, which encodes the plastid-localized cysteine-rich DnaJ protein, regulates carotenoid accumulation and abiotic stress resistance in various plant species.Ectopic expression of Or promotes carotenoid accumulation in various crop species [17][18][19][20][21][22][23][24][25].In sweetpotato, the IbOr protein functions as a holdase chaperone, a post-transcriptional regulator of phytoene synthase (PSY), a rate-limiting enzyme in the carotenoid biosynthetic pathway [21,26].Furthermore, IbOr interacts with oxygen-evolving enhancer protein 2-1 (PsbP) in the chloroplast, a member of the protein complex in photosystem II (PSII), and protects it from heat-induced denaturation [26,27].IbOr also interacts with carotenoid cleavage dioxygenase 4 (IbCCD4) in the chloroplast, which is consistent with the location of the interaction of IbOr with IbPSY and IbPsbP.The interaction of IbOr and IbCCD4 does not affect the cleavage of β-carotene [28].Thus, IbOr is a multifunctional protein that exhibits strong holdase chaperone activity and contributes to carotenoid accumulation and abiotic stress tolerance in plants.
Recently, a dominant or allele carrying a single nucleotide polymorphism (SNP; referred to as the golden SNP) was identified in melon (Cucumis melo); this SNP causes arginine (Arg) to histidine (His) substitution in the CmOr protein and is associated with high β-carotene accumulation in melon [18].Overexpression of AtOr-His, a variant of the Arabidopsis thaliana or gene carrying the golden SNP, in Arabidopsis calli and tomato (Solanum lycopersicum), leads to high-level accumulation of carotenoids [22,29].In addition, the other SNP was identified between low-and high-carotenoid genotypes in carrot (Daucus carota); this transition of T to C results in a change of the codon TTG to TCG, causing SNP of leucine (Leu) to serine (Ser) and is related to high β-carotene accumulation in carrot [30].Recently, we performed site-directed mutagenesis of the IbOr gene, leading to Arg to His substitution at position 96 (based on the CmOr protein) in the IbOr protein (IbOr-R96H), and generated IbOr-R96H transgenic sweetpotato calli.Transgenic calli overexpressing IbOr-R96H showed significantly higher carotenoid contents and enhanced salt and heat stress tolerance than those overexpressing the wild-type IbOr (IbOr-WT) gene [31].In this study, we generated transgenic sweetpotato plants overexpressing IbOr-R96H to study its effect at the whole-plant level.Our results showed that overexpression of IbOr-R96H increased the content of total carotenoids, especially β-carotene, in storage roots of transgenic sweetpotato plants.In addition, we investigated abiotic stress tolerance assay using leaf discs and monitored transcript levels of carotenoid biosynthesis and degradation genes.Interestingly, IbOr-R96H transgenic plants exhibited high DPPH radical scavenging activity in both fresh storage roots and leaves compared with non-transgenic (NT) and IbOr-WT plants.

Plant Materials and Growth Conditions
A white-fleshed sweetpotato cultivar, Xushu 29, was kindly provided by the Sweetpotato Research Institute, Chinese Academy of Agricultural Science, Xuzhou, China.Plants were grown in a growth chamber under controlled conditions (25 ± 3 °C temperature, under a light intensity of 150 μmol m −2 s −1 and 16 h light/8 h dark photoperiod).To analyze carotenoid content, the storage roots of sweetpotato plants were harvested 4 months after planting.

Plasmid Construction and Agrobacterium-Mediated Transformation
Site-directed mutagenesis of IbOr was performed as described previously [31].IbOr-WT (accession no.HQ828087) and IbOr-R96H were cloned into the pGWB5 binary Gateway vector under the control of the cauliflower mosaic virus (CaMV) 35S promoter to generate fusions with the GFP gene.The constructs were transformed into sweetpotato embryogenic calli using Agrobacterium tumefaciens strain GV3101 [23].

RNA Extraction and qRT-PCR Analysis of Carotenogenesis-Related Genes
Total RNA was extracted from the leaves and storage roots of 3-week-old and 4month-old transgenic sweetpotato plants, respectively, using the method reported previously [31].To analyze the expression of sweetpotato genes involved in carotenoid biosynthesis and degradation pathways, qRT-PCR was performed on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), according to the manufacturer's instructions, using EvaGreen fluorescent dye and gene-specific primers (Tables S1  and S2).

Analysis of Carotenoid Contents
Carotenoids were extracted from storage roots of 4-month-old transgenic sweetpotato plants using acetone (0.01% BHT).The extracted carotenoids were analyzed using the Agilent 1260 high-performance liquid chromatography (HPLC) system (Hewlett-Packard, Waldbronn, Germany), as described previously [32].All extraction procedures were conducted under dim light to avoid pigment degradation and loss.Carotenoid contents were expressed as μg g −1 dry weight (DW) of storage roots.

Analysis of Heat Tolerance
To induce heat stress, leaf discs (12 mm in diameter) were excised from the 3rd, 4th, and 5th leaves of 1-month-old transgenic and NT sweetpotato plants, and exposed to high temperature (47 °C) in the dark for 12 h.To visualize the degree of damage caused by heat stress, heat-treated leaf discs were floated on 3,3′-diaminobenzidine (DAB) solution (1 mg mL −1 in water; pH 3.8), as described previously [27].To measure the extent of cellular damage or membrane disruption, ion leakage was quantified using an ion conductivity meter (MTD, Schwerzenbach, Switzerland).

Determination of ABA Content
ABA was extracted from the leaf discs of individual plants and its amount was determined with a plant hormone abscisic acid (ABA) ELISA Kit (MyBioSource Inc., San Diego, CA, USA) following manufacturer instruction.Absorbance at 450 nm was detected using Bio-Rad i-Mark Microplate Reader (Bio-Rad).Three independent experiments were performed.

Antioxidant Activity Analysis
The analysis of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity is a widely used spectrophotometric methods for the assessment of the antioxidant capacities inhibiting lipid oxidation in complex biological mixtures such as plant or food extracts.The assay was analyzed as described previously [33].Leaf and storage root extracts of transgenic and NT sweetpotato plants were prepared in methanol, and the absorbance of the extracts was determined at 517 nm using a spectrophotometer.A calibration curve was constructed using L-Ascorbic acid (AsA) as a standard, and the DPPH radical scavenging activity of the extracts was calculated as AsA equivalents per gram of tested samples.

Statistical Analysis
Data were subjected to one-way analysis of variance (ANOVA).Significant differences were identified by Tukey's honestly significant difference (HSD) post hoc test at p < 0.05.

IbOr-R96H Overexpression Induces Overaccumulation of Carotenoids in Storage Roots
Previously, overexpression of IbOr, AtOr, and SbOr carrying the golden SNP increased the total carotenoid and β-carotene contents of various plant species.Additionally, IbOr-R96H transgenic calli accumulated significantly higher levels of total carotenoids and β-carotene than IbOr-WT transgenic calli, based on the color of the callus [22,31].To assess whether the color difference correlated with carotenoid accumulation, we analyzed the carotenoid content of storage roots and leaves of NT, IbOr-WT, and IbOr-R96H plants by HPLC.The IbOr-R96H transgenic plants exhibited differences in the carotenoid composition and content of storage roots (Figure 3 and Table S3) and leaves (Table S4) compared with NT and IbOr-WT transgenic plants.Depending on the transgenic line, the total carotenoid contents of IbOr-R96H and IbOr-WT transgenic storage roots were 5.4-19.6-and1.7-3.2-foldhigher, respectively, than that of NT storage roots.Among various carotenoids, β-carotene showed a dramatic overaccumulation in IbOr-R96H transgenic storage roots; its level was 21.7-186.2-foldhigher in IbOr-R96H transgenic storage roots than in NT storage roots.The amount of α-carotene varied from 0.09-0.37μg g −1 DW in storage roots of the three IbOr-R96H transgenic lines; however, α-carotene was undetectable in NT and IbOr-WT transgenic storage roots (Figure 3).Interestingly, β-cryptoxanthin content varied from 0.05 μg g −1 DW (line #9) to 0.13 μg g −1 DW (line #1) in IbOr-R96H transgenic storage roots, but was undetectable in NT and IbOr-WT storage roots (Figure 3 and Table S3).Thus, the promotion of β-carotene accumulation in IbOr-R96H transgenic plants was consistent with that observed in IbOr-R96H transgenic sweetpotato calli [31].In leaves, lutein was the most abundant carotenoid; this result was obtained not only in NT plants but also in transgenic lines (three IbOr-WT and three IbOr-R96H).Unlike storage roots, the total carotenoid and β-carotene contents of IbOr-WT and IbOr-R96H plants were slightly lower than those of NT plants (Table S4).

IbOr-R96H Overexpression Significantly Altered the Expression of Carotenoid Metabolismrelated Genes
Previously, we showed that most carotenoid biosynthetic pathway genes were upregulated in the transgenic sweetpotato calli and plants overexpressing IbOr [23,31,33].To determine whether the overaccumulation of carotenoids in storage roots of IbOr-R96H plants is correlated with altered expression of carotenogenesis-related genes, we analyzed the transcript levels of carotenoid biosynthesis genes in NT, IbOr-WT, and IbOr-R96H plants by qRT-PCR.The results showed that the Or gene was highly expressed in IbOr-R96H transgenic storage roots.In addition, phytoene synthase (PSY), phytoene desaturase (PDS), zeta-carotene desaturase (ZDS), lycopene ε-cyclase (LCY-ε), and beta-carotene hydroxylase (CHY-β) genes were expressed to higher levels in IbOr-WT and IbOr-R96H transgenic storage roots than in NT storage roots (Figure 4).We previously showed that expression levels of carotenoid cleavage genes were elevated in IbOr-R96H transgenic calli compared with NT calli [31].Therefore, we investigated the transcript levels of carotenoid degradation pathway genes in IbOr-R96H storage roots.Expression levels of carotenoid cleavage dioxygenases 4 (CCD4) and 9-cis-epoxycarotenoid dioxygenase (NCED), which are involved in carotenoid cleavage, were higher in IbOr-WT and IbOr-R96H transgenic storage roots than in NT storage roots, consistent with our previous results.However, the transcript levels of these genes were lower in IbOr-R96H storage roots than in IbOr-WT storage roots.In addition, the expression levels of carotenoid degradation genes such as CCD1, carotene isomerase (D27), lycopene cleavage dioxygenase (LCD), and aldehyde dehydrogenase (ADH), which are involved in lycopene and β-carotene cleavage, were lower in IbOr-R96H transgenic storage roots than in NT and IbOr-WT transgenic storage roots (Figure 5).These results indicate that enhanced accumulation of carotenoids in IbOr-R96H transgenic storage roots might be an effect of the lower expression of carotenoid cleavage genes.

IbOr-R96H Transgenic Plants Exhibit Increased Antioxidant Activity and Heat Stress Tolerance
Previously, IbOr-WT transgenic sweetpotato calli exhibited increased antioxidant activity and salt stress tolerance.Furthermore, IbOr-R96H transgenic calli also showed enhanced tolerance to salt and heat stresses [31,33].To investigate the antioxidant activity in transgenic sweetpotato, we analyzed the DPPH radical scavenging activity in fresh storage roots and leaves of NT, IbOr-WT, and IbOr-R96H plants (Figure 6).In storage roots, the DPPH radical scavenging activity was 5.5-10.2-and1.9-3.0-foldhigher in IbOr-R96H and IbOr-WT transgenic plants, respectively, than in NT plants (Figure 6A).Additionally, in contrast to the normal (no stress) condition, the DPPH activity in IbOr-R96H leaves was up to 1.85-fold higher than that in NT leaves after 12 h of heat stress treatment (Figure 6B).Previously, transgenic IbOr-WT and IbOr-R96H calli exhibited increased tolerance to heat stress compared with NT calli [31].To compare the heat stress tolerance of transgenic and NT sweetpotato plants, leaf discs prepared from NT, IbOr-WT, and IbOr-R96H plants were incubated at 47 °C for 12 h, and membrane damage was examined by DAB staining and ion leakage analysis.NT plants showed severe membrane damage, as evident from the dark-brown coloration of leaf discs, whereas IbOr-WT and IbOr-R96H leaf discs displayed less membrane damage.IbOr-R96H transgenic plants showed greater heat stress tolerance than NT and IbOr-WT plants (Figure 7A).After 12 h of heat stress treatment, leaves of NT and IbOr-WT plants showed 66% and 42.8-43.8%ion leakage, respectively.By contrast, leaves of IbOr-R96H plants exhibited significantly less ion leakage (21.2-23.75%)at the same time point (Figure 7B).In addition, to investigate the relationship between greater heat stress tolerance and the contents of abscisic acid (ABA), we quantified ABA contents in plants under normal growth conditions and after exposure to heat stress.Under normal and after 6 h of heat stress conditions, ABA contents were higher in IbOr-WT and IbOr-R96H plants than in NT plants.This result is consistent with the findings that the expression level of NCED, a key enzyme in ABA biosynthesis, was higher in IbOr-WT and IbOr-R96H transgenic plants, as shown in Figure 5.After 12 h of heat stress treatment, the highest values of ABA contents were recorded in NT plants compared to IbOr-WT and IbOr-R96H plants (Figure 7C).These results suggest that the increases in DPPH radical scavenging activity and ABA contents in IbOr-R96H plants lead to greater tolerance to heat stress.

Discussion
Overexpression of the golden SNP-carrying mutant CmOr allele resulted in enhanced carotenoid content and orange-colored melon fruit phenotypes [18].Overexpression of the IbOr gene carrying the same mutation resulted in high total carotenoid and β-carotene contents and enhanced abiotic stress tolerance in sweetpotato [31].In this study, we successfully generated transgenic sweetpotato plants overexpressing IbOr-R96H.In carrot, the root color depends on the contents of different carotenoids: orange-colored carrot genotypes contain mostly α-carotene and β-carotene; yellow and red carrots contain lutein and lycopene, respectively; and white genotypes contain almost no carotenoid [34].In storage roots of sweetpotato, the β-carotene content varies greatly with the color of different varieties of sweetpotato, which ranges from white to light yellow, deep yellow, and dark orange [35,36].Overexpression of IbOr-R96H in sweetpotato changed the color of storage roots from white to orange (Figure 2).These results suggest that overexpression of IbOr-R96H increased the contents of carotenoids, especially β-carotene, in storage roots.
Carotenoids play important roles in the primary and secondary metabolism of plants and accumulate to high levels in tissues of some plants.Xylem and phloem surrounding the cambium layers represent secondary tissues in the plant vascular system.Xylem conducts water, while phloem conducts amino acids and sucrose.Interestingly, overexpression of IbOr-R96H in sweetpotato changed the color of the area surrounding the cambium ring in storage roots (Figure 2B, right).Unlike most crops, the phloem and xylem tissues in carrot roots are visually well separated by the vascular cambium.Previously, some studies showed that the level of carotene was higher in secondary phloem than in secondary xylem of carrot roots.In phloem chromoplasts, carotene is mainly deposited in the crystalline form.Carotenoid metabolites induce structural changes in carotene deposited in chromoplasts, the main organelles of carotenoid accumulation, and crystalline chromoplast development leads to the massive accumulation of carotene pigments [37][38][39][40].Both carrot and sweetpotato contain high levels of β-carotene and α-carotene.In the carrot root, the proportion of crystalline chromoplasts is the highest compared with other substructures.However, in sweetpotato, chromoplasts exert various substructures such as globular, crystals, and plastoglobuli, together with membrane substructures [41].Therefore, it is possible that a color change in storage roots of IbOr-WT and IbOr-R96H transgenic sweetpotato plants, specifically in the area surrounding the cambium ring, is related to the overaccumulation of carotenoids and structural modification of carotenes.
In addition, storage roots of IbOr-R96H plants accumulated carotenoids to higher levels than IbOr-WT and NT plants.Similar to IbOr-R96H transgenic sweetpotato calli, storage roots of IbOr-R96H plants showed a dramatic increase in total carotenoid content (up to 19.6-fold) and β-carotene content (up to 186.2-fold) compared with those of NT plants.Additionally, the total carotenoid and β-carotene contents of IbOr-R96H storage roots were 3.2-6.1-and11.5-99.3-foldgreater than those of IbOr-WT storage roots, respectively (Figure 3).In contrast to the results obtained with storage roots, the leaves of NT, IbOr-WT, and IbOr-R96H plants showed no significant differences in carotenoid contents (Table S4).Unlike non-photosynthetic tissues such as fruits and roots, leaves rarely exhibit an overaccumulation of carotenoids.In plant leaves, carotenoids are continuously synthesized and degraded to maintain optimal photosynthesis through carotenoid steady-state regulatory mechanisms [42][43][44][45].These results indicate that leaves maintain carotenoid homeostasis, however, through unknown mechanisms.
Carotenoid cleavage reactions play a major role in determining the carotenoid content of plants.Lycopene, β-carotene, and zeaxanthin act as precursors of important apocarotenoids.The conversion of carotenoids into apocarotenoids is catalyzed by carotenoid cleavage dioxygenases (CCDs), which target different double bonds of the carotenoid polyene chain.The 9-cis-epoxycarotenoid dioxygenase (NCEDs) cleave 9-cis-violaxanthin and 9cis-neoxanthin into xanthoxin, and synthesize abscisic acid (ABA), an important hormone involved in plant stress response and seed maturation.The lycopene cleavage dioxygenase (LCD) enzyme, along with ADH, converts lycopene into bixin, a polyene.Enzymes including CCD1, D27, LCD, and ADH mediate lycopene and β-carotene cleavage [46][47][48].We showed that overexpression of IbOr-R96H in sweetpotato storage roots increased not only the total carotenoid content but also CCD4 and NCED expression levels (Figure 4).In addition, the expression levels of carotenoid degradation genes such as CCD1, D27, LCD, and ADH were lower in storage roots of IbOr-R96H plants than in those of NT and IbOr-WT plants (Figure 5).These results indicate that the down-regulation of genes encoding carotenoid cleavage enzymes leads to carotenoid overaccumulation in storage roots of IbOr-R96H plants compared with those of NT and IbOr-WT plants.Interestingly, we found various expression patterns among CCDs in IbOr-R96H transgenic sweetpotato plants.Especially, we found that there is an inverse effect between CCD1 and CCD4.While expression levels of CCD1 was lower than in NT storage roots, CCD4 is highly expressed (Figure 5).Similar to CCD1, loss of function in CCD4 induces carotenoid accumulation in plants.However, in sweetpotato, IbCCD4 interacts specifically with IbOr and its interaction did not affect β-carotene degradation [28].The relation between CCD4 expression and carotenoid accumulation in IbOr-WT and IbOr-R96H plants remains to be studied in detail.
IbOr stabilizes IbPSY, a key regulator of the carotenoid biosynthetic pathway, via its chaperone activity under heat stress.Overexpression of IbOr in sweetpotato calli and plants improved tolerance to heat stress [26,30].Here, we showed that leaf discs excised from IbOr-R96H transgenic sweetpotato plants displayed greater heat stress tolerance than IbOr-WT and NT plants.Until 6 h of heat stress conditions, ABA contents were higher in IbOr-WT and IbOr-R96H plants than in NT plants (Figure 7).In addition, the DPPH radical scavenging activity, which reflects the antioxidant activity, was higher in IbOr-R96H plants than in IbOr-WT and NT plants (Figure 6).Carotenoids as antioxidants can quench singlet oxygen and scavenge superoxide anion radicals.In addition, DPPH is widely used to evaluate the ability of compounds as a free radical scavenger to enhance antioxidant activity [49,50].Furthermore, ABA is an important isoprenoid stress-induced phytohormone.The ABA is quickly synthesized and is regulated by the ABA biosynthesis NCED genes under abiotic stresses such as temperature, drought and salinity [51,52].In this study, DPPH radical activity and ABA contents with enhanced transcript level of NCED in transgenic lines were much higher than those in the NT line.These results suggest that overexpression of IbOr-R96H in sweetpotato not only increases the contents of total carotenoid and β-carotene but also enhances heat stress tolerance via increased ABA level and DPPH radical scavenging activity.The relation between heat stress tolerance and ABA contents in IbOr-WT and IbOr-R96H plants remains to be studied in detail.
To further analyze the regulatory mechanisms of IbOr-R96H, we are currently investigating the potential IbOr-R96H-interacting partners associated with carotenoid overaccumulation and abiotic stress tolerance.Furthermore, we anticipate that site-specific mutagenesis of IbOr combined with CRISPR-Cas9-mediated base-editing techniques could be an effective tool for the improvement of carotenoid contents in sweetpotato.Therefore, we are currently under-developing transgenic sweetpotato plants expressing IbOr-R96H.Overall, the IbOr-R96H gene could help us cope with global food and nutrition security problems, and help to establish a sustainable society in the face of climate change.

Conclusions
In this study, we showed that overexpression of IbOr-R96H significantly increased carotenoid accumulation and antioxidant activity in sweetpotato storage roots to levels higher than those achieved by the overexpression of IbOr-WT.Storage roots of IbOr-R96H transgenic sweetpotato plants showed up to 19.6-and 186.2-fold higher total carotenoid and β-carotene contents, respectively, compared with those of NT plants.In addition, gene expression analysis indicated that genes encoding carotenoid cleavage enzymes might be involved in the overaccumulation of carotenoids in IbOr-R96H storage roots.Furthermore, at 47 °C, IbOr-R96H transgenic sweetpotato plants displayed greater heat stress tolerance than IbOr-WT plants, which was associated with higher DPPH radical scavenging activity and ABA contents.Taken together, our results demonstrate that overexpression of IbOr-R96H in sweetpotato confers heat stress tolerance by triggering the production of carotenoids and improving free radical scavenging capabilities.

Figure 1 .
Figure 1.Generation of transgenic sweetpotato plants overexpressing IbOr-WT and IbOr-R96H.(A) Schematic representation of the constructs used for the production of transgenic sweetpotato plants overexpressing IbOr-WT and IbOr-R96H.(B) Genotyping transgenic plants using IbOr-specific primers.PC, positive control.(C) Transcript levels of IbOr in transgenic sweetpotato plants.Data represent the mean ± standard deviation (SD) of three technical replicates.To determine whether the overexpression of IbOr-R96H increased carotenoid synthesis in transgenic sweetpotato plants, we first evaluated the morphological phenotypes of NT, IbOr-WT, and IbOr-R96H plants.Stem cuttings of NT, IbOr-WT (#2, #9, #10), and IbOr-R96H (#1, #9, #10) plants were grown in plastic pots for 4 months.One-month-old plants of transgenic IbOr-WT and IbOr-R96H lines exhibited no significant phenotypic alterations in aerial plant parts compared with NT plants (Figure 2A).However, the skin of IbOr-R96H transgenic storage roots exhibited a light-red color compared with that of NT and IbOr-WT storage roots (Figure 2B, left).Interestingly, storage roots of IbOr-R96H plants exhibited a light-orange color overall and a dark-orange color around the cambium rings, whereas storage roots of IbOr-WT plants showed a deeper yellow color around the cambium layers than those of NT plants (Figure 2B, right).

Figure 3 .
Figure 3. Quantitative HPLC analysis of total carotenoids and individual carotenoid compounds in storage roots of NT and transgenic sweetpotato plants.Data represent mean ± SD of three independent replicates.Significant differences between IbOr-WT, IbOr-R96H, and NT plants are indicated with asterisks ** p < 0.01; ANOVA, followed by Tukey's HSD post hoc test).

Figure 6 .
Figure 6.Antioxidant activity in transgenic sweetpotato plants.(A,B) Analysis of DPPH radical scavenging activity in the storage roots (A) and leaves (B) of NT, IbOr-WT, and IbOr-R96H plants treated with heat stress for 12 h.Data represent mean ± SD of three independent replicates.Significant differences between IbOr-WT, IbOr-R96H, and NT plants are indicated with asterisks (* p < 0.05, ** p < 0.01; one-way ANOVA, followed by Tukey's HSD post hoc test).

Figure 7 .
Figure 7. Analysis of heat stress tolerance in transgenic sweetpotato plants using leaf discs.(A) Images of leaf discs and quantification of ROS production in leaves using DAB staining.(B) Ion leakage from detached leaves treated with heat for 0, 6, and 12 h.(C) Quantitative analysis of abscisic acid (ABA) contents in leaves under heat stress.Data represent mean ± SD of three independent replicates.Significant differences between IbOr-WT, IbOr-R96H, and NT plants are indicated with asterisks (* p < 0.05, ** p < 0.01; one-way ANOVA, followed by Tukey's HSD post hoc test).