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Sweetpotato (Ipomoea batatas(L.) Lam) is the world’s seventh most important food crop by production quantity. Cultivated sweetpotato is a hexaploid (2n = 6x = 90), and its genome (B1B1B2B2B2B2) is quite complex due to polyploidy, self-incompatibility, and high heterozygosity. Here we established a haploid-resolved and chromosome-scalede novoassembly of autohexaploid sweetpotato genome sequences. Before constructing the genome, we created chromosome-scale genome sequences inI. trifidausing a highly homozygous accession, Mx23Hm, with PacBio RSII and Hi-C reads. Haploid-resolved genome assembly was performed for a sweetpotato cultivar, Xushu18 by hybrid assembly with Illumina paired-end (PE) and mate-pair (MP) reads, 10X genomics reads, and PacBio RSII reads. Then, 90 chromosome-scale pseudomolecules were generated by aligning the scaffolds onto a sweetpotato linkage map.De novoassemblies were also performed for chloroplast and mitochondrial genomes inI. trifidaand sweetpotato. In total, 34,386 and 175,633 genes were identified on the assembled nucleic genomes ofI. trifidaand sweetpotato, respectively. Functional gene annotation and RNA-Seq analysis revealed locations of starch, anthocyanin, and carotenoid pathway genes on the sweetpotato genome. This is the first report of chromosome-scalede novoassembly of the sweetpotato genome. The results are expected to contribute to genomic and genetic analyses of sweetpotato.

Lignin is associated with cell wall rigidity, water and solute transport, and resistance to diverse stresses in plants. Lignin consists of polymerized monolignols (p-coumaryl, coniferyl, and sinapyl alcohols), which are synthesized by cinnamyl alcohol dehydrogenase (CAD) in the phenylpropanoid pathway. We previously investigated cold-induced IbCAD1 expression by transcriptome profiling of cold-stored tuberous roots of sweetpotato (Ipomoea batatas [L.] Lam). In this study, we confirmed that IbCAD1 expression levels depended on the sweetpotato root type and were strongly induced by several abiotic stresses. We generated transgenic sweetpotato plants overexpressing IbCAD1 (TC plants) to investigate CAD1 physiological functions in sweetpotato. TC plants displayed lower root weights and lower ratios of tuberous roots to pencil roots than non-transgenic (NT) plants. The lignin contents in tuberous roots of NT and TC plants differed slightly, but these differences were not significant. By contrast, monolignol levels and syringyl (S)/guaiacyl (G) ratios were higher in TC plants than NT plants, primarily owing to syringyl unit accumulation. Tuberous roots of TC plants displayed enhanced low-temperature (4 °C) storage with lower malondialdehyde and H2O2 contents than NT plants. We propose that high monolignol levels in TC tuberous roots served as substrates for increased peroxidase activity, thereby enhancing antioxidation capacity against cold stress–induced reactive oxygen species. Increased monolignol contents and/or increased S/G ratios might contribute to pathogen-induced stress tolerance as a secondary chilling-damage response in sweetpotato. These results provide novel information about CAD1 function in cold stress tolerance and root formation mechanisms in sweetpotato.

Class III peroxidases are involved in plant responses to various environmental conditions. Peroxidase production is triggered by various stimuli and activates stress-related signaling pathways to help plants cope with abiotic and biotic stresses. Recent evidence suggests that the interplay between diverse peroxidases plays a critical role in regulating stress responses. However, the underlying molecular mechanisms remain poorly understood. Previous research showed that transgenic sweetpotato overexpressing the swpa4 peroxidase gene exhibited increased tolerance to oxidative stress and high salinity. In this study, loss of function of the swpa4 peroxidase gene in sweetpotato was assessed in transgenic plants with constitutive RNAi-mediated suppression of the swpa4 gene. Compared with control plants, leaf discs from RNAi-suppressed lines exhibited increased damage following treatment with the oxidative stressors hydrogen peroxide and methyl viologen. Transgenic plants also showed reduced resistance to high salinity conditions compared with control plants. Repression of swpa4 also led to reduced expression of the endogenous stress-related peroxidase genes swpa1, swpa2, and swpn1. These findings suggest that swpa4 regulates signaling pathways involved in the regulation of peroxidase metabolism during the stress response in sweetpotato.

Sweetpotato (Ipomoea batatas [L.] Lam) is an important starch crop that ensures food and nutrition security in the era of climate change. Sweetpotato is tolerant to environmental stresses such as drought, high temperature, and high salt, and therefore, is well adapted to marginal lands; however, it is relatively vulnerable to flooding stress, which severely reduces its yield and commercial value. To understand the flooding stress response of sweetpotato, we performed comparative transcriptome analysis of the leaves of two sweetpotato cultivars with contrasting flooding stress tolerance levels: Yeonjami (YJM; flooding tolerant) and Jeonmi (JM; flooding sensitive). Both cultivars were partially submerged in water for 0, 0.5, and 3 days. RNA-seq data of both cultivars revealed 14,229 differentially expressed genes (DEGs), which were categorized into seven clusters and six groups, based on the expression pattern of co-expressed DEGs and expression duration of DEGs, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that DEGs of distinguishing functions between the two cultivars were involved in plant hormone signaling, carbohydrate transport, and mitogen-activated protein kinase (MAPK) signaling. Based on these results, we predict that YJM promotes adventitious growth, whereas JM exhibits shoot elongation under flooding stress. The expression levels of several key candidate genes involved in flooding tolerance correlated well with the comparative transcriptomics data. Overall, this study provides further insights into the molecular mechanism of flooding stress response in sweetpotato, and reveals candidate genes that could be used for developing new flooding tolerant sweetpotato cultivars.

Sweetpotato (Ipomoea batatas [L.] Lam) is one of the most important food crops, as it ensures food and nutrition security in the face of climate change, given its high carbohydrate, antioxidant (carotenoid), dietary fiber, and mineral contents. The Orange protein of sweetpotato (IbOr) shows high molecular chaperone activity. Previously, we showed that transgenic sweetpotato plants overexpressing the IbOr gene exhibited strong tolerance to abiotic stresses such as high temperature (47 °C) and drought, owing to the increased contents of carotenoids including β-carotene. Interestingly, the expression of the sweetpotato carotenoid cleavage dioxygenase 4 (IbCCD4) gene was also significantly up-regulated in IbOr-overexpressing sweetpotato plants. In this study, we confirmed that the IbCCD4 gene is highly expressed in carotenoid-rich leaves and storage roots of three types of sweetpotato cultivars, each with a different storage root color (pale yellow, orange, and purple). The IbCCD4 protein showed interaction with IbOr in yeast two-hybrid and pull-down assays. Additionally, in vitro, carotenoid cleavage assays indicated that β-carotene is the major substrate of IbCCD4. Interestingly, the interaction between IbCCD4 and IbOr did not affect β-carotene degradation. Further characterization of the IbCCD4 gene in transgenic sweetpotato plants will provide new insights into carotenoid homeostasis under severe stress conditions.

We previously reported that transgenic Arabidopsis thaliana plants overexpressing the sweet potato peroxidase gene swpa4 under the control of the cauliflower mosaic virus (CaMV) 35 s promoter showed increased levels of reactive oxygen species (ROS) and nitric oxide (NO), and higher expression of ROS and NO related genes than control plants. Here, we investigated the effect of swpa4 overexpression on the abiotic and biotic stress tolerance levels of Arabidopsis plants. Methyl viologen (MV) treatment-induced oxidative stress caused visible damage to the seedlings and rosette leaves of all Arabidopsis plants, although the symptoms were more severe in control plants than in transgenic lines. Additionally, survival rates and ion leakage showed a slight decline in transgenic lines but a more severe decline in control plants after MV treatment. Transgenic plants also showed enhanced tolerance to drought stress. Dehydration treatment, followed by rehydration, resulted in a greater change in the relative water content and lipid peroxidation of control plants than in that of transgenic lines. However, transgenic plants did not show enhanced resistance to biotic stresses such as bacterial and fungal pathogens. These results indicate that transgenic Arabidopsis plants can efficiently regulate defense levels during oxidative stress via the overexpression of swpa4.

In plants, the orange (Or) gene plays roles in regulating carotenoid biosynthesis and responses to environmental stress. The present study investigated whether the expression of rice Or (OsOr) gene could enhance rice tolerance to heat stress conditions. The OsOr gene was cloned and constructed with OsOr or OsOr-R115H (leading to Arg to His substitution at position 115 on the OsOr protein), and transformed into rice plants. The chlorophyll contents and proline contents of transgenic lines were significantly higher than those of non-transgenic (NT) plants under heat stress conditions. However, we found that the levels of electrolyte leakage and malondialdehyde in transgenic lines were significantly reduced compared to NT plants under heat stress conditions. In addition, the levels of expression of four genes related to reactive oxygen species (ROS) scavenging enzymes (OsAPX2, OsCATA, OsCATB, OsSOD-Cu/Zn) and five genes (OsLEA3, OsDREB2A, OsDREB1A, OsP5CS, SNAC1) responded to abiotic stress was showed significantly higher in the transgenic lines than NT plants under heat stress conditions. Therefore, OsOr-R115H could be exploited as a promising strategy for developing new rice cultivars with improved heat stress tolerance.

Environmental stress leads to the production of reactive oxygen species (ROS) and consequently oxidative stress, which limits plant productivity. Thus, new crop cultivars that are able to overcome oxidative stress are required to address the global issues of environmental, food, and energy security. Sweetpotato (Ipomoea batatas [L.] Lam) is considered an emerging multifunctional crop because of its high carbohydrate content and adaptation to marginal lands. Moreover, sweetpotato offers several health benefits for the aging population, as it is rich in low-molecular-weight antioxidants (LMWAs) (e.g., carotenoids, ascorbate, tocopherols, and polyphenols), dietary fiber, potassium, and minerals. We speculate that metabolic engineering of LMWAs in sweetpotato will lead to the development of smart cultivars with enhanced levels of antioxidants and abiotic stress tolerance. This review describes the current status of metabolic engineering of LMWAs in sweetpotato and the potential of sweetpotato in ensuring food and nutrition security in the twenty-first century. Here, we focus on carotenoids, ascorbate, tocopherols, and polyphenols including anthocyanins in transgenic sweetpotato plants. Additionally, we discuss the potential role of the sweetpotato Orange (IbOr) gene, involved in carotenoid accumulation and stress tolerance, in improving crop productivity under the changing global climate and alleviating the problem of vitamin A deficiency. Genetic engineering of the biosynthetic and metabolic genes of LMWAs in sweetpotato is currently under investigation via molecular breeding. We predict that metabolic engineering of antioxidants in sweetpotato will lead to the development of new cultivars with enhanced nutritional value and abiotic stress tolerance, which will ensure global food and nutrition security in the face of climate change.

Global climate change is expected to cause progressively increased frequency and severity of drought events, which further seriously limit plant growth and crop yields. Increasing water use efficiency (WUE) and yield per unit rainfall are one of the most important challenges in dry land agriculture. Here, we reviewed the comprehensive technical strategies including conserving water to combine both increased agricultural productivity and resource conservation; enquiring into how crop plants respond to drought through morphological, physiological, and molecular modifications that occur in all plant organs; breeding for drought tolerance where there is a delineated stress environment and genotype × environment interactions are stable; effective conservation of rainfall and high efficiency of use. In addition, we discussed the preponderance of biological water-saving measures, which embraces improvements in WUE and drought tolerance, by genetic improvement and physiological regulation. Sustainable agriculture would be benefited from modern engineering such as biological engineering, conservation tillage, and breeding technologies.

The ERF (ethylene-responsive element-binding factor) transcription factor family plays important roles in abiotic and biotic stress responses in plants. An EAR motif-containing ERF gene, IbERF4, was isolated from sweetpotato [Ipomoea batatas (L.) Lam]. IbERF4 was highly expressed in the early stage of storage roots and can be induced by various stresses. By overexpressing IbERF4 in Arabidopsis, the transgenic Arabidopsis showed greater sensitivity to drought and salt stress by down-regulating stress-related genes, such as rd22, rd29, AtCBF1 and AtCBF2. Overexpressing IbERF4 in sweetpotato made the transgenic sweetpotato more sensitive to salt stress and made propagating new seedlings more difficult. Consistently, the transcriptomic profiling results showed that genes involved in oxidative stress were significantly down-regulated in sweetpotato overexpression lines. These results indicated that IbERF4 plays as key regulators in sweetpotato abiotic stress.

Secretory class III peroxidases are involved in plant responses to a range of biological stress conditions, including oxidative stress. To investigate the stress-related functions of the swpa4 peroxidase gene in sweetpotato (Ipomoea batatas L.), transgenic plants overexpressing the swpa4 gene under the control of the CaMV 35S promoter were generated using Agrobacterium-mediated transformation. Peroxidase activity was 3- to 13-fold higher in transgenic lines than in control plants. Transgenic plants were tested for tolerance to stress. Following treatment with 400 mM hydrogen peroxide (H2O2), leaf discs from transgenic plants showed approximately 13–26% less damage than control plants. Transgenic plants also showed enhanced tolerance to high salinity conditions. Following treatment with NaCl, photosynthetic capacity and total chlorophyll contents were less severely impacted in swpa4 transgenic plants than in control plants. Aerial plant parts and storage root yields were not significantly different between transgenic and control plants following cultivation in field conditions. These results indicate that transgenic sweetpotato lines were able to respond efficiently to oxidative and saline stress via overexpression of swpa4.

Invertase inhibitor (INH) post-translationally regulates the activity of invertase, which hydrolyzes sucrose into glucose and fructose, and plays essential roles in plant growth and development. However, little is known about the role of INH in growth and responses to environmental challenges in sweetpotato. Here, we identified and characterized an INH-like gene (IbINH) from sweetpotato. IbINH belongs to the pectin methylesterase inhibitor super family. IbINH transcript was the most abundant in storage roots. IbINH mRNA levels were significantly up-regulated in response to drought, abscisic acid (ABA), salicyclic acid (SA) and jasmonic acid (JA) treatments. Overexpressing IbINH in sweetpotato (SI plants) led to the decrease of plant growth and the increase of drought tolerance, while down-regulation of IbINH (RI plants) by RNAi technology resulted in vigorous growth and drought sensitivity. Furthermore, sucrose was increased and hexoses was decreased in SI plants, but the opposite results were observed in RI plants. Moreover, higher levels of sugars were accumulated in SI plants in comparison to non-transgenic plants (NT plants) and RI plants during water deficit. In addition, ABA biosynthesis-involved and abiotic stress response-involved genes were prominently up-regulated in SI plants under drought stress. Taken together, these results indicate that IbINH mediates plant growth and drought stress tolerance in sweetpotato via induction of source-sink strength and ABA-regulated pathway.

Dehydroascorbate reductase (DHAR) plays a critical role in the regeneration of ascorbic acid (AsA), and widely involved in plant tolerance to biotic and abiotic stresses. In this study, the IbDHAR3 gene was cloned from sweetpotato cultivar Xushu 18 by RT-PCR. The full-length of this gene was 813 bp which encodes 270 amino acids. The IbDHAR3 protein contained two conserved domains of glutathione S-transferase (GST) and GST-C-DHAR, and one chloroplast transit peptide with 52 amino acids length. Transient expression in tobacco leaf epidermal cells indicated that IbDHAR3 protein is subcellular localized to chloroplast. The qRT-PCR results revealed that the relative expression level of IbDHAR3 in leaves is much higher than that in other tissues, and could be up-regulated by ABA, drought, salinity, and high-temperature stresses. The seed germination rate and root elongation were increased in contrast to wild type under mannitol and NaCl stresses in T3 transgenic Arabidopsis overexpressing IbDHAR3 gene. The soil drought experiments showed that the overexpression of IbDHAR3 gene in Arabidopsis reduced the malondialdehyde (MDA) and the H2O2 content, enhanced the level of AtGR gene expression, superoxide dismutase (SOD), ascorbate peroxidase (APX), DHAR activity, and the AsA content. Therefore, overexpression of IbDHAR3 gene could enhance the ability of scavenging reactive oxygen species such as H2O2 by promoting AsA-glutathione cycle and related antioxidant enzymes system, thereby contributing to increased stress tolerance in Arabidopsis.

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.

In Arabidopsis, the cytosolic redox protein thioredoxin h2 (Trx-h2) is anchored to the cytoplasmic endomembrane through the myristoylated second glycine residue (Gly2). However, under cold stress, the cytosolic Trx-h2 is rapidly translocated to the nucleus, where it interacts with and reduces the cold-responsive C-repeat-binding factors (CBFs), thus activating cold-responsive (COR) genes. In this study, we investigated the significance of fatty acid modification of Trx-h2 under cold conditions by generating transgenic Arabidopsis lines in the trx-h2 mutant background, overexpressing Trx-h2 (Trx-h2OE/trx-h2) and its point mutation variant Trx-h2(G/A) [Trx-h2(G/A)OE/trx-h2], in which the Gly2 was replaced by alanine (Ala). Due to the lack of Gly2, Trx-h2(G/A) was incapable of myristoylation, and a part of Trx-h2(G/A) localized to the nucleus even under warm temperature. As no time is spent on the demyristoylation and subsequent nuclear translocation of Trx-h2(G/A) under a cold snap, the ability of Trx-h2(G/A) to protect plants from cold stress was greater than that of Trx-h2. Additionally, COR genes were up-regulated earlier in Trx-h2(G/A)2OE/trx-h2 plants than in Trx-h2OE/trx-h2 plants under cold stress. Consequently, Trx-h2(G/A)2OE/trx-h2 plants showed greater cold tolerance than Col-0 (wild type) and Trx-h2OE/trx-h2 plants. Overall, our results clearly demonstrate the significance of the demyristoylation of Trx-h2 in enhancing plant cold/freezing tolerance.

Sweetpotato (Ipomoea batatas [L.] Lam) is a prospective food crop that ensures food and nutrition security under the dynamic changes in global climate. Peroxidase (POD) is a multifunctional enzyme involved in diverse plant physiological processes, including stress tolerance and cell wall lignification. Although various POD genes were cloned and functionally characterized in sweetpotato, the role of POD in lignification and low-temperature storage ability of sweetpotato tuberous roots is yet to be investigated. In this study, we isolated the cold-induced lignin forming peroxidase (IbLfp) gene of sweetpotato, and analyzed its physiological functions. IbLfp showed more predominant expression in fibrous roots than in other tissues. Moreover, IbLfp expression was up-regulated in leaves and roots under cold stress, and was altered by other abiotic stresses. Tuberous roots of transgenic sweetpotato lines overexpressing IbLfp (LP lines) showed improved tolerance to low temperature, with lower malondialdehyde and hydrogen peroxide contents than non-transgenic sweetpotato plants under cold stress. The enhanced cold tolerance of LP lines could be attributed to the increased basal activity of POD, which is involved in reactive oxygen species (ROS) scavenging. Moreover, greater accumulation of lignin could also contribute to the enhanced cold tolerance of LP lines, as lignin acts as a protective barrier against invading pathogens, which is a secondary symptom of chilling injury in sweetpotato. Overall, the results of this study enhance our understanding of the function of POD in low-temperature storage of sweetpotato tuberous roots.

Tocopherols are lipid-soluble compounds regarded as vitamin E compounds and they function as antioxidants in scavenging lipid peroxyl radicals and quenching reactive oxygen species (ROS). In our previous studies, we isolated five tocopherol biosynthesis genes from sweetpotato (Ipomoea batatas [L.] Lam) plants including 4-hydroxyphenylpyruvate dioxygenase (IbHPPD). HPPD is the first regulatory enzyme in vitamin E biosynthesis and serves to catalyze in the first steps α-tocopherol and plastoquinone biosynthesis by converting 4-hydroxyphenylpyruvate (HPP) to homogentisic acid (HGA). In this study, we generated transgenic sweetpotato plants overexpressing IbHPPD under the control of cauliflower mosaic virus (CaMV) 35S promoter (referred to as HP plants) via Agrobacterium-mediated transformation to understand the function of IbHPPD in sweetpotato. Three transgenic lines (HP3, HP14 and HP15) with high transcript levels of IbHPPD were selected for further characterization. Compared with non-transgenic (NT) plants, HP plants exhibited enhanced tolerance to multiple environmental stresses, including salt, drought, and oxidative stresses. In addition, HP plants showed increased tolerance to the herbicide sulcotrione, which is involved in the inhibition of the HPPD. Interestingly, after stress treatments, HP plants also showed higher abscisic acid (ABA) contents than NT plants. Under dehydrated condition, HP plants displayed an elevated α-tocopherol content to 19-27% in leaves compared with NT plants. These results indicate that increased abiotic stress tolerance in HP plants is related to inducing enhancement of α-tocopherol and ABA contents.

Sweetpotato (Ipomoea batatas [L.] Lam) is an economically important, nutrient- and pigment-rich root vegetable used as both food and feed. Root-knot nematode (RKN), Meloidogyne incognita, causes major yield losses in sweetpotato and other crops worldwide. The identification of genes and mechanisms responsible for resistance to RKN will facilitate the development of RKN resistant cultivars not only in sweetpotato but also in other crops. In this study, we performed RNA-seq analysis of RKN resistant cultivars (RCs; Danjami, Pungwonmi and Juhwangmi) and susceptible cultivars (SCs; Dahomi, Shinhwangmi and Yulmi) of sweetpotato infected with M. incognita to examine the induced and constitutive defense response-related transcriptional changes. During induced defense, genes related to defense and secondary metabolites were induced in SCs, whereas those related to receptor protein kinase signaling and protein phosphorylation were induced in RCs. In the uninfected control, genes involved in proteolysis and biotic stimuli showed differential expression levels between RCs and SCs during constitutive defense. Additionally, genes related to redox regulation, lipid and cell wall metabolism, protease inhibitor and proteases were putatively identified as RKN defense-related genes. The root transcriptome of SCs was also analyzed under uninfected conditions, and several potential candidate genes were identified. Overall, our data provide key insights into the transcriptional changes in sweetpotato genes that occur during induced and constitutive defense responses against RKN infection.

Sweetpotato (Ipomoea batatas L. Lam) is an economically important crop that is cultivated for its storage roots. Storage roots provide a source of valuable nutrients, processed foods, animal feeds, and pigments. Sweetpotato storage roots spoil during post-harvest handling because of wounding, which makes them more susceptible to disease-causing microorganisms. Curing to promote wound healing is a common method to control microbial spoilage during post-harvest storage. However, molecular mechanisms underlying the process of curing in sweetpotato storage roots are unknown. To better understand the biology behind curing, the transcriptome of the sweetpotato cultivar, Pungwonmi, was studied using RNA-seq. Storage roots of sweetpotato were treated at 33 °C (Curing) and 13 °C (Control) for 3 days. RNA-seq data identified 78,781 unigenes and 3,366 differentially expressed genes by over log2 fold change (FC) 2 and-2. During curing, DEGs encoded genes related to drought/salt stress responses, phyto-hormones (e.g., auxin, ethylene and jasmonic acid), and proteolysis, were up-regulated, whereas those related to redox state, phyto-hormones (e.g., salicylic acid and brassinosteroids), and lignin and flavonoid biosynthesis were down-regulated. Additionally, among the candidate genes, DEGs encoded genes related to proteolysis and pathogen defense, such as protease inhibitors and lipid transfer proteins, were highly up-regulated during curing and storage. This study provides a valuable resource to further understand the molecular basis of curing-mediated wound healing in sweetpotato storage roots. Moreover, genes revealed in this work could present targets for the development of sweetpotato varieties with improved post-harvest storage characteristics.

IbOr-R96H resulted in carotenoid overaccumulation and enhanced abiotic stress tolerance in transgenic sweetpotato calli. The Orange (Or) protein is involved in the regulation of carotenoid accumulation and tolerance to various environmental stresses. Sweetpotato IbOr, with strong holdase chaperone activity, protects a key enzyme, phytoene synthase (PSY), in the carotenoid biosynthetic pathway and stabilizes a photosynthetic component, oxygen-evolving enhancer protein 2-1 (PsbP), under heat and oxidative stresses in plants. Previous studies of various plant species demonstrated that a single-nucleotide polymorphism (SNP) from Arg to His in Or protein promote a high level of carotenoid accumulation. Here, we showed that the substitution of a single amino acid at position 96 (Arg to His) of wild-type IbOr (referred to as IbOr-R96H) dramatically increases carotenoid accumulation. Sweetpotato calli overexpressing IbOr-WT or IbOr-Ins exhibited 1.8- or 4.3-fold higher carotenoid contents than those of the white-fleshed sweetpotato Yulmi (Ym) calli, and IbOr-R96H overexpression substantially increased carotenoid accumulation by up to 23-fold in sweetpotato calli. In particular, IbOr-R96H transgenic calli contained 88.4-fold higher levels of β-carotene than those in Ym calli. Expression levels of carotenogenesis-related genes were significantly increased in IbOr-R96H transgenic calli. Interestingly, transgenic calli overexpressing IbOr-R96H showed increased tolerance to salt and heat stresses, with similar levels of malondialdehyde to those in calli expressing IbOr-WT or IbOr-Ins. These results suggested that IbOr-R96H is a useful target for the generation of efficient industrial plants, including sweetpotato, to cope with growing food demand and climate change by enabling sustainable agriculture on marginal lands.

Previously transgenic tobacco plants over-expressing the sweet potato peroxidase gene swpa4 exhibited increased tolerance to various stress conditions. Expression of swpa4 in tobacco also has a positive effect on expression of genes related to reactive oxygen species (ROS) and nitric oxide (NO). Expression levels of respiratory burst oxidase protein D (RBOHD), a major gene regulating ROS, and nitric oxide associated 1 (NOA1), a gene involved in NO generation, are significantly affected by peroxidase inhibitor treatments. In this study, swpa4 was expressed in the rbohD and noa1 knock-out mutants of Arabidopsis, as well as in wild-type plants. The transgenic plants showed increased levels of ROS and NO. Expression of swpa4 activated expression of genes related to ROS and NO. This suggested that swpa4 regulated H2O2 and NO homeostasis, thereby establishing a potential molecular link between the defense signaling pathways mediated by ROS and NO.

Sweetpotato [Ipomoea batatas (L.) Lam] is an important crop used for food, animal feed, and production of industrial materials. Although it is adapted to a wide range of unfavorable conditions, including drought and high salt, sweetpotato is vulnerable to low temperature, making it difficult to cultivate in low temperature regions. To understand the molecular responses occurring in sweetpotato leaves under low temperature stress, de novo transcriptome assembly was performed in leaves under low temperature stress (LT) and during recovery (RC). In comparison with non-treated controls (NT), 2461 and 1017 differentially expressed genes (DEGs) were identified in LT and RC leaves, respectively. When expression in RC and LT samples was directly compared, 2053 DEGs were detected. To increase understanding of the DEGs, the three datasets were analyzed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genome (KEGG) database. The CBF transcriptional cascade, a well-known cold response pathway, was investigated using transcriptomic analysis. In contrast with reports from the cold-tolerant Arabidopsis thaliana, none of the COR genes identified in sweetpotato showed increased expression in response to low temperature. Genes involved in antioxidant enzyme pathways mediating responses to reactive oxygen species (ROS) were investigated during low temperature response. This work provides insight into the molecular basis of the responses of sweetpotato to cold stress. This increased understanding of gene regulation in response to cold stress in sweetpotato will be beneficial for future research into molecular-assisted breeding.