Overexpression of Arabidopsis P3B increases heat and low temperature stress tolerance in transgenic sweetpotato.

BACKGROUND: Sweetpotato (Ipomoea batatas [L.] Lam) is suitable for growth on marginal lands due to its abiotic stress tolerance. However, severe environmental conditions including low temperature pose a serious threat to the productivity and expanded cultivation of this crop. In this study, we aimed to develop sweetpotato plants with enhanced tolerance to temperature stress. RESULTS: P3 proteins are plant-specific ribosomal P-proteins that act as both protein and RNA chaperones to increase heat and cold stress tolerance in Arabidopsis. Here, we generated transgenic sweetpotato plants expressing the Arabidopsis ribosomal P3 (AtP3B) gene under the control of the CaMV 35S promoter (referred to as OP plants). Three OP lines (OP1, OP30, and OP32) were selected based on AtP3B transcript levels. The OP plants displayed greater heat tolerance and higher photosynthesis efficiency than wild type (WT) plants. The OP plants also exhibited enhanced low temperature tolerance, with higher photosynthesis efficiency and less membrane permeability than WT plants. In addition, OP plants had lower levels of hydrogen peroxide and higher activities of antioxidant enzymes such as peroxidase and catalase than WT plants under low temperature stress. The yields of tuberous roots and aerial parts of plants did not significantly differ between OP and WT plants under field cultivation. However, the tuberous roots of OP transgenic sweetpotato showed improved storage ability under low temperature conditions. CONCLUSIONS: The OP plants developed in this study exhibited increased tolerance to temperature stress and enhanced storage ability under low temperature compared to WT plants, suggesting that they could be used to enhance sustainable agriculture on marginal lands.

Overexpression of the IbMYB1 gene in an orange-fleshed sweet potato cultivar produces a dual-pigmented transgenic sweet potato with improved antioxidant activity.

The R2R3-type protein IbMYB1 is a key regulator of anthocyanin biosynthesis in the storage roots of sweet potato [Ipomoea batatas (L.) Lam]. Previously, we demonstrated that IbMYB1 expression stimulated anthocyanin pigmentation in tobacco leaves and Arabidopsis. Here, we generated dual-pigmented transgenic sweet potato plants that accumulated high levels of both anthocyanins and carotenoids in a single sweet potato storage root. An orange-fleshed cultivar with high carotenoid levels was transformed with the IbMYB1 gene under the control of either the storage root-specific sporamin 1 (SPO1) promoter or the oxidative stress-inducible peroxidase anionic 2 (SWPA2) promoter. The SPO1-MYB transgenic lines exhibited higher anthocyanin levels in storage roots than empty vector control (EV) or SWPA2-MYB plants, but carotenoid content was unchanged. SWPA2-MYB transgenic lines exhibited higher levels of both anthocyanin and carotenoids than EV plants. Analysis of hydrolyzed anthocyanin extracts indicated that cyanidin and peonidin predominated in both overexpression lines. Quantitative reverse transcription-polymerase chain reaction analysis demonstrated that IbMYB1 expression in both IbMYB1 transgenic lines strongly induced the upregulation of several genes in the anthocyanin biosynthetic pathway, whereas the expression of carotenoid biosynthetic pathway genes varied between transgenic lines. Increased anthocyanin levels in transgenic plants also promoted the elevation of proanthocyanidin and total phenolic levels in fresh storage roots. Consequently, all IbMYB1 transgenic plants displayed much higher antioxidant activities than EV plants. In field cultivations, storage root yields varied between the transgenic lines. Taken together, our results indicate that overexpression of IbMYB1 is a highly promising strategy for the generation of transgenic plants with enhanced antioxidant capacity.

Structural characteristics of low-digestible sweet potato starch prepared by heat-moisture treatment.

The objectives of this study were to optimize the conditions for the preparation of sweet potato starch containing a high proportion of slowly digestible starch (SDS) by heat-moisture treatment (HMT), and to investigate the structural properties of SDS and resistant starch (RS) fractions isolated from this modified starch. Response surface methodology evaluation of considering temperature, time, and moisture content was conducted to determine the HMT condition for maximizing SDS yield. Maximum SDS content (50.9%) was obtained after 8.6 h at 113 °C with 22.8% moisture level. Scanning electron micrographs of RS fractions displayed growth ring structure and hollow regions. X-ray diffraction patterns of SDS + RS and RS fractions were not changed by HMT. Gelatinization enthalpy of the SDS + RS fraction was decreased more than that of the RS fraction. The results indicated that the SDS fraction consisted of less perfect crystallites and amorphous regions, whereas the RS fraction was composed mostly of crystalline regions.

Phylogenetic Placement and Morphological Characterization of Sclerotium rolfsii (Teleomorph: Athelia rolfsii) Associated with Blight Disease of Ipomoea batatas in Korea.

In this study, we aimed to characterize fungal samples from necrotic lesions on collar regions observed in different sweetpotato growing regions during 2015 and 2016 in Korea. Sclerotia appeared on the root zone soil surface, and white dense mycelia were observed. At the later stages of infection, mother roots quickly rotted, and large areas of the plants were destroyed. The disease occurrence was monitored at 45 and 84 farms, and 11.8% and 6.8% of the land areas were found to be infected in 2015 and 2016, respectively. Fungi were isolated from disease samples, and 36 strains were preserved. Based on the cultural and morphological characteristics of colonies, the isolates resembled the reference strain of Sclerotium rolfsii. Representative strains were identified as S. rolfsii (teleomorph: Athelia rolfsii) based on phylogenetic analysis of the internal transcribed spacer and large subunit genes along with morphological observations. To test the pathogenicity, sweetpotato storage roots were inoculated with different S. rolfsii strains. 'Yulmi' variety displayed the highest disease incidence, whereas 'Pungwonmi' resulted in the least. These findings suggested that morphological characteristics and molecular phylogenetic analysis were useful for identification of S. rolfsii.

Suppression of the ß-carotene hydroxylase gene increases ß-carotene content and tolerance to abiotic stress in transgenic sweetpotato plants.

ß-carotene, a carotenoid that plays a key photo-protective role in plants is converted into zeaxanthin by ß-carotene hydroxylase (CHY-ß). Previous work showed that down-regulation of IbCHY-ß by RNA interference (RNAi) results in higher levels of ß-carotene and total carotenoids, as well as salt stress tolerance, in cultured transgenic sweetpotato cells. In this study, we introduced the RNAi-IbCHY-ß construct into a white-fleshed sweetpotato cultivar (cv. Yulmi) by Agrobacterium-mediated transformation. Among the 13 resultant transgenic sweetpotato plants (referred to as RC plants), three lines were selected for further characterization on the basis of IbCHY-ß transcript levels. The RC plants had orange flesh, total carotenoid and ß-carotene contents in storage roots were 2-fold and 16-fold higher, respectively, than those of non-transgenic (NT) plants. Unlike storage roots, total carotenoid and ß-carotene levels in the leaves of RC plants were slightly increased compared to NT plants. The leaves of RC plants also exhibited tolerance to methyl viologen (MV)-mediated oxidative stress, which was associated with higher 2,2-diphenyl-1- picrylhydrazyl (DPPH) radical-scavenging activity. In addition, RC plants maintained higher levels of chlorophyll and higher photosystem II efficiency than NT plants after 250 mM NaCl stress. Yield of storage roots did not differ significantly between RC and NT plants. These observations suggest that RC plants might be useful as a nutritious and environmental stress-tolerant crop on marginal lands around the world.

Preparation of slowly digestible sweet potato Daeyumi starch by dual enzyme modification.

Sweet potato Daeyumi starch was dually modified using glycogen branching enzyme (BE) from Streptococcus mutans and amylosucrase (AS) from Neisseria polysaccharea to prepare slowly digestible starch (SDS). Dually modified starches had higher SDS and resistant starch (RS) contents than control starch. The branched chain length distributions of the BE-modified starches indicated an increase in short side-chains [degree of polymerization (DP)≤12] compared with native starch. AS treatment of the BE-modified starches decreased the proportion of short side-chains and increased the proportion of long side-chains (DP≥25) and molecular mass. It also resulted in a B-type X-ray diffraction pattern and an increased relative crystallinity. Regarding thermal properties, the BE-modified starches showed no endothermic peak, whereas the BEAS-modified starches had a broader melting temperature range and lower melting enthalpy compared to native starch. The combined enzymatic treatment resulted in novel glucan polymers with slow digestion properties.

Comparative analysis of phytochemicals and polar metabolites from colored sweet potato (Ipomoea batatas L.) tubers.

We determined the phytochemical diversity, including carotenoids, flavonoids, anthocyanins, and phenolic acids, in sweet potatoes (Ipomoea batatas L.) with distinctive flesh colors (white, orange, and purple) and identified hydrophilic primary metabolites. Carotenoid content was considerably higher in orange-fleshed sweet potatoes, wherein ß-carotene was the most plentiful, and anthocyanins were detected only in purple-fleshed sweet potatoes. The levels of phenolic acids and flavonoids were relatively higher in purple-fleshed sweet potatoes than those in the other two varieties. Forty-one primary and 18 secondary metabolite profiles were subjected to multivariate statistical analyses, which fully distinguished among the varieties and separated orange- and purple-fleshed sweet potatoes from white-fleshed sweet potatoes based on the high levels of sugars, sugar alcohols, and secondary metabolites. This is the first study to determine comprehensive metabolic differences among different color-fleshed sweet potatoes and provides useful information for genetic manipulation of sweet potatoes to influence primary and secondary metabolism.

Enhanced accumulation of carotenoids in sweetpotato plants overexpressing IbOr-Ins gene in purple-fleshed sweetpotato cultivar.

Sweetpotato [Ipomoea batatas (L.) Lam] is an important root crop that produces low molecular weight antioxidants such as carotenoids and anthocyanin. The sweetpotato orange (IbOr) protein is involved in the accumulation of carotenoids. To increase the levels of carotenoids in the storage roots of sweetpotato, we generated transgenic sweetpotato plants overexpressing IbOr-Ins under the control of the cauliflower mosaic virus (CaMV) 35S promoter in an anthocyanin-rich purple-fleshed cultivar (referred to as IbOr plants). IbOr plants exhibited increased carotenoid levels (up to 7-fold) in their storage roots compared to wild type (WT) plants, as revealed by HPLC analysis. The carotenoid contents of IbOr plants were positively correlated with IbOr transcript levels. The levels of zeaxanthin were ∼ 12 times elevated in IbOr plants, whereas ß-carotene increased ∼ 1.75 times higher than those of WT. Quantitative RT-PCR analysis revealed that most carotenoid biosynthetic pathway genes were up-regulated in the IbOr plants, including PDS, ZDS, LCY-ß, CHY-ß, ZEP and Pftf, whereas LCY-ɛ was down-regulated. Interestingly, CCD1, CCD4 and NCED, which are related to the degradation of carotenoids, were also up-regulated in the IbOr plants. Anthocyanin contents and transcription levels of associated biosynthetic genes seemed to be altered in the IbOr plants. The yields of storage roots and aerial parts of IbOr plants and WT plants were not significantly different under field cultivation. Taken together, these results indicate that overexpression of IbOr-Ins can increase the carotenoid contents of sweetpotato storage roots.

Exogenous sucrose utilization and starch biosynthesis among sweet potato cultivars.

Three sweetpotato cultivars were investigated for their starch content and amylose/amylopectin ratio. Ym starch contains 87.2% amylopectin and 12.8% amylose, when total starch was calculated as 100%. The Zm cultivar contains 33.6% amylopectin and 18.2% amylose, and its total starch was calculated as 51.8% of that of Ym. The Hm cultivar contains 39.1% amylopectin and 30.5% amylose, and its total starch was 69.6%. We analyzed the expression levels of starch and sucrose biosynthesis-related genes including AGPases a, b, and c; sucrose synthases I and II; starch synthase I; GBSS I; and SBEs I and II. All genes tested in this experiment were detected only in Ym, while several genes showed very faint or no expression in Zm and Hm. We also measured tissue-specific expression of these genes in whole plants of Ym. Most of the genes are expressed in the stem and roots of the plants. Expression profiles of starch synthesis-related genes of the sweetpotato leaves were investigated after supplementing the different concentrations of sucrose solution. All genes in Ym were clearly induced by sucrose, but the expression levels of some of these genes did not change in Zm and Hm. The total starch content of Ym, Zm, and Hm gradually increased over time on addition of 3%, 6%, and 9% sucrose concentrations. The greatest accumulation was observed in Ym at 48h, and it was almost 2.24 times higher than that of the (0%) control, while Zm and Hm showed 1.76 and 1.91 times higher levels of starch, respectively. These results indicate that cooperative expression of all related genes is essential for starch biosynthesis from sucrose. This is the first report on different sucrose contents and the efficiency with which exogenous sucrose switches on gene expression of starch biosynthesis-related genes among cultivars.

Ethanol and water extract of purple sweet potato exhibits anti-atherosclerotic activity and inhibits protein glycation.

It is well known that extracts of purple sweet potato (PSP) have potent antioxidant activity. However, it has not been established whether extracts of PSP inhibit oxidation of low-density lipoprotein (LDL) or protein glycation. LDL oxidation and protein glycation are well-known risk factors for chronic metabolic diseases, such as atherosclerosis and diabetes mellitus. Chopped and sliced PSP and yellow sweet potato (YSP) were extracted individually at a concentration of 1 g of PSP tuber/mL using either ethanol or water for 6 hours. The PSP ethanol extract (100-fold diluted) showed stronger radical (2,2-diphenyl-1-picrylhydrazyl radical) scavenging activity than the water extract of PSP and the ethanol extract of YSP (up to a sixfold higher activity). The ethanol extract of PSP also exhibited the highest increase in ferric reducing ability among all extracts. Cupric ion-mediated LDL oxidation was strongly inhibited by the ethanol extract of PSP, with similar potency to vitamin C treatment (final concentration, 10 mM). The PSP extract strongly inhibited fructose-mediated protein glycation as determined by fluorescence spectroscopy. The PSP extract-treated apolipoprotein (apo) A-I showed a decreased multimerization pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas glycated apoA-I showed the strongest multimeric band. PSP extract treatment also inhibited the uptake of oxidized LDL into human macrophage cells with suppression of malondialdehyde production in the cell culture medium. In conclusion, these results suggest that the extract of PSP can be used as a putative anti-atherosclerotic and antidiabetic agent with strong antioxidant functions. This is the first report to show the biological functions of PSP extract to treat hyperlipidemic and hyperglycemic disorders.