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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.

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.

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.

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.

Auxin is a major plant hormone that regulates many developmental processes in plants. Arabidopsis thaliana YUCCA6 (AtYUCCA6) mediates the tryptophan-dependent auxin biosynthesis pathway in A. thaliana. To study the effect of auxin in sweetpotato [Ipomoea batatas (L.) Lam], we generated transgenic sweetpotato plants expressing AtYUCCA6 under the control of the oxidative stress-inducible SWPA2 promoter (referred to as SY plants). SY plants displayed high-auxin phenotypic traits such as narrow downward-curled leaves, increased height, and increased node numbers. SY plants showed increased tolerance to 5 μM of methyl viologen-mediated oxidative stress by maintaining a lower ion leakage than wild-type (WT) plants. Moreover, SY plants exhibited higher relative water contents and lower malondialdehyde contents than WT plants under drought stress. Young SY plants also showed inhibition of adventitious root growth. SY plants showed significantly lower storage root formation than WT plants. Our results indicate that AtYUCCA6-mediated auxin overproduction is associated with tolerance to oxidative and drought stress. It also suggests that negatively regulates the formation of storage roots in sweetpotato.

Trehalose-6-phosphate synthase (TPS) plays an important regulatory role in the response of plants to multiple abiotic stresses. However, our knowledge of the stress tolerance functions of TPS genes in sweet potato (Ipomoea batatas [L.] Lam) remains limited. In the present study, we isolated and functionally characterized the sweet potato gene encoding TPS1, IbTPS1. Sequence analysis showed that IbTPS1 belongs to class I TPS proteins and harbors highly conserved acceptor (glucose-6-phosphate [Glc6P]) and donor (uridine diphosphoglucose [UDP-Glc]) binding sites. The IbTPS1 gene showed the highest level of constitutive expression in leaves and storage roots of sweet potato plants and was induced by several environmental stresses including drought, salt, and heat shock. The IbTPS1 protein might be localized to the cytosol. Complementation assay of yeast tps1Δ and tps1Δtps2Δ growth-defective mutants confirmed the TPS activity of IbTPS1, and truncation of the N-terminal extension of IbTPS1 (ΔNIbTPS1) increased the catalytic activity of the protein. Additionally, expression of IbTPS1 in yeast conferred abiotic stress tolerance to dehydration, salinity, and oxidation, and expression of ΔNIbTPS1 further enhanced the tolerance to abiotic stresses. The results of this study advance our understanding of the functions of IbTPS1 under abiotic stress conditions. Our results suggest that the IbTPS1 gene is an excellent candidate for improving the stress tolerance of different crop plants.

The ethylene-responsive element binding factor (ERF) family is a large family of transcription factors involved in plant development and environmental stress responses. We previously reported the identification of 29 putative substrates of Mitogen-activated Protein Kinase3 (AtMPK3), AtMPK4 and AtMPK6, based on a solid-phase phosphorylation screening using a lambda phage expression library in Arabidopsis thaliana. In this study, a putative MPK substrate, AtERF72 (At3g16770), was strongly phosphorylated by AtMPK6 on the serine residue at position 151 (Ser151). AtERF72 binds to the GCC box (AGCCGCC) in the promoters of several pathogenesis-related (PR) genes and activates their transcription. We also show that the DNA-binding activity of AtERF72 is enhanced upon phosphorylation by AtMPK6 in vitro. In addition, transient co-expression experiments in Arabidopsis protoplasts revealed that effector constructs expressing a mutant variant of AtERF72, AtERF72S151D (carrying a Ser to aspartic acid [Asp] substitution at amino acid position 151) showed higher expression of the β-glucuronidase (GUS) reporter gene driven by the GCC box element than effector constructs expressing the wild-type AtERF72. Furthermore, yeast two-hybrid assays revealed that the interaction between AtERF72S151D and TGA4/OBF4 was stronger than that between wild-type AtERF72 and TGA4/OBF4. Since AtERF72S151D is equivalent to AtERF72 phosphorylated by AtMPK6 at Ser151, these results suggest that the phosphorylation of AtERF72 by AtMPK6 triggers an event of transcriptional regulation from defence signalling in Arabidopsis.

Sweetpotato [Ipomoea batatas (L.) Lam] serves as a sustainable food source and ensures nutrition security in the face of climate change. Recently, farmers have developed increased interest in replacing rice with sweetpotato in paddy fields for higher income. However, sweetpotato is more susceptible to flooding stress than other abiotic stresses including drought and salinity. Here, we selected flooding tolerant sweetpotato cultivars based on biochemical characterization. Young seedlings of 33 sweetpotato cultivars were subjected to flooding stress for 20 days, and Yeonjami (YJM) was identified as the most flooding tolerant sweetpotato cultivar. Plant growth and biochemical characteristics of YJM were compared with those of Jeonmi (JM), a flooding sensitive sweetpotato cultivar. Under flooding stress, YJM showed higher content of chlorophyll and lower inhibition of plant height and fibrous root length than JM. Biochemical characterization revealed that although malondialdehyde and hydrogen peroxide contents were increased in fibrous roots of both cultivars, the amount of increase was 4-fold lower in YJM than in JM. Additionally, leaves of YJM showed higher ascorbate peroxidase activity than those of JM under flooding stress. Our results suggest that high membrane stability and antioxidant capacity are important flooding tolerance factors in sweetpotato. Furthermore, several flooding tolerance-related genes involved in starch and sucrose metabolism, fermentation, and cell wall loosening showed earlier induction and higher transcript levels in YJM leaves and fibrous roots than in JM tissues under flooding stress. Thus, phenotypic and biochemical characterization suggests that YJM could be used as a flooding tolerant sweetpotato cultivar.

Late embryogenesis abundant (LEA) proteins are small, highly hydrophilic proteins that act as protectors of macromolecules and increase abiotic stress tolerance in plants. We previously reported that overexpressing sweetpotato IbLEA14 under the control of the CaMV 35S promoter increased osmotic and salt stress tolerance in transgenic sweetpotato calli. In this study, we generated transgenic poplar plants (Populus alba × P. glandulosa) expressing IbLEA14 under the control of the oxidative stress-inducible SWPA2 promoter (referred to as SL plants). Among the 15 SL plants obtained, three lines (SL2, SL7, and SL12) were established based on IbLEA14 transcript levels, tolerance to salt stress and Southern blot analysis. The SL plants exhibited less damage in response to methyl viologen-mediated oxidative stress than non-transgenic (NT) plants. SL plants also showed enhanced tolerance to drought, salt, and heat stress, which was associated with higher photosystem II efficiency and lower malondialdehyde levels compared with NT plants. Furthermore, SL plants had higher levels of monolignol biosynthesis-related gene transcripts under drought stress compared with NT plants. Finally, SL plants exhibited increased tolerance to heat stress, which is associated with the high thermostability of IbLEA14 protein. SL plants might be useful for reforestation on global marginal lands, including desertification and reclaimed areas.

Sweetpotato [Ipomoea batatas (L.) Lam] represents an attractive starch crop that can be used to facilitate solving global food and environmental problems in the 21st century. It can be used as industrial bioreactors to produce various high value-added materials, including bio-ethanol, functional feed, antioxidants, as well as food resources. The non-profit Center for Science in the Public Interest (CSPI) announced sweetpotato as one of the ten ‘super foods’ for better health, since it contains high levels of low molecular weight antioxidants such as vitamin-C, vitamin-E and carotenoids, as well as dietary fiber and potassium. The United States Department of Agriculture (USDA) also reported that sweetpotato is the best bioenergy crop among starch crops on marginal lands, that does not affect food security. The Food and Agriculture Organization (FAO) estimated that world population in 2050 will be 9.7 billion, and require approximately 1.7 times more food than today. In this respect, sweetpotato will be a solution to solving problems such as food, energy, health, and environment facing the globe in the 21st century. In this paper, the current status of resources, and cultivation of sweetpotato in the world was first described. Development of a new northern route of the sweetpotato and its prior tasks of large scale cultivation of sweetpotato, were also described in terms of global food security, and production of high-value added biomaterials.

Dramatic increase in global population accompanied by rapid industrialization in developing countries has led to serious environmental, food, energy, and health problems. The Food and Agriculture Organization of the United Nations has estimated world population will increase to 9.7 billion by 2050 and require approximately 1.7 times more food, and more than 3.5 times energy than that of today. Particularly, sweetpotato is easy to cultivate in unfavorable conditions such as heat, drought, high salt, and marginal lands. In this respect, sweetpotato is an industrially valuable starch crop. To replace crops associated with these food and energy problems, it is necessary to develop new crops with improved nutrients and productivity, that can be grown on marginal lands, including desertification areas using plant biotechnology. For this purpose, exploring useful genes and developing genetically modified crops are essential strategies. Currently, sweetpotato [Ipomoea batatas (L.) Lam.] have been re-evaluated as the best health food and industrial crop that produces starch and low molecular weight antioxidants, such as vitamin A, vitamin E, anthocyanins and carotenoids. This review will focus on the current status of research on sweetpotato biotechnology on omics including genome sequencing, transcriptome, proteomics and molecular breeding. In addition, prospects on molecular breeding of sweetpotato on marginal lands for sustainable development were described.

Carotenoids play essential roles in various light-harvesting processes in plants and help protect the photosynthetic machinery from photo-oxidative damage. Orange genes, which play a role in carotenoid accumulation, have recently been isolated from several plant species, and their functions have been intensively investigated. The Orange gene (IbOr) of sweet potato [Ipomoea batatas (L.) Lam] helps maintain carotenoid homeostasis to improve plant tolerance to environmental stress. IbOr, a protein with strong holdase chaperone activity, directly interacts with phytoene synthase, a key enzyme involved in carotenoid biosynthesis, in plants under stress conditions, resulting in increased carotenoid accumulation and abiotic stress tolerance. In addition, IbOr interacts with the oxygen-evolving enhancer protein 2-1, a member of a protein complex in photosystem II that is denatured under heat stress. Transgenic sweet potato plants overexpressing IbOr showed enhanced tolerance to high temperatures (47 °C). These findings indicate that IbOr protects plants from environmental stress not only by controlling carotenoid biosynthesis, but also by directly stabilizing photosystem II. In this review, we discuss the functions of IbOr and Or proteins in other plant species and their possible biotechnological applications for molecular breeding for sustainable development on marginal lands.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-1087-2) contains supplementary material, which is available to authorized users.

Oxidative stress caused by reactive oxygen species (ROS) under various environmental stresses significantly reduces plant productivity. Tocopherols (collectively known as vitamin E) are a group of lipophilic antioxidants that protect cellular components against oxidative stress. Previously, we isolated five tocopherol biosynthesis genes from sweetpotato (Ipomoea batatas [L.] Lam) plants, including tocopherol cyclase (IbTC). In this study, we generated transgenic sweetpotato plants overexpressing IbTC under the control of cauliflower mosaic virus (CaMV) 35S promoter (referred to as TC plants) via Agrobacterium-mediated transformation to understand the function of IbTC in sweetpotato. Three transgenic lines (TC2, TC9, and TC11) with high transcript levels of IbTC were selected for further characterization. High performance liquid chromatography (HPLC) analysis revealed that α-tocopherol was the most predominant form of tocopherol in sweetpotato tissues. The content of α-tocopherol was 1.6-3.3-fold higher in TC leaves than in non-transgenic (NT) leaves. No significant difference was observed in the tocopherol content of storage roots between TC and NT plants. Additionally, compared with NT plants, TC plants showed enhanced tolerance to multiple environmental stresses, including salt, drought, and oxidative stresses, and showed consistently higher levels of photosystem II activity and chlorophyll content, indicating abiotic stress tolerance. These results suggest IbTC as a strong candidate gene for the development of sweetpotato cultivars with increased α-tocopherol levels and enhanced abiotic stress tolerance.

The abscisic acid (ABA)-responsive element binding factors (ABFs) play important regulatory roles in multiple abiotic stresses responses. However, information on the stress tolerance functions of ABF genes in sweetpotato (Ipomoea batatas [L.] Lam) remains limited. In the present study, we isolated and functionally characterized the sweetpotato IbABF4 gene, which encodes an abiotic stress-inducible basic leucine zipper (bZIP) transcription factor. Sequence analysis showed that the IbABF4 protein contains a typical bZIP domain and five conserved Ser/Thr kinase phosphorylation sites (RXXS/T). The IbABF4 gene was constitutively expressed in leaf, petiole, stem, and root, with the highest expression in storage root body. Expression of IbABF4 was induced by ABA and several environmental stresses including drought, salt, and heat shock. The IbABF4 protein localized to the nucleus, exhibited transcriptional activation activity, and showed binding to the cis-acting ABA-responsive element (ABRE) in vitro. Overexpression of IbABF4 in Arabidopsis thaliana not only increased ABA sensitivity but also enhanced drought and salt stress tolerance. Furthermore, transgenic sweetpotato plants (hereafter referred to as SA plants) overexpressing IbABF4, generated in this study, exhibited increased tolerance to drought, salt, and oxidative stresses on the whole plant level. This phenotype was associated with higher photosynthetic efficiency and lower malondialdehyde and hydrogen peroxide content. Levels of endogenous ABA content and ABA/stress-responsive gene expression were significantly upregulated in transgenic Arabidopsis and sweetpotato plants compared with wild-type plants under drought stress. Our results suggest that the expression of IbABF4 in Arabidopsis and sweetpotato enhances tolerance to multiple abiotic stresses through the ABA signaling pathway.

IbSPF1, a novel target of IbMPK3/IbMPK6, regulates biotic stress response in sweetpotato. Environmental stresses due to biotic and abiotic factors negatively affect crop quality and productivity. To minimize the damage caused by these factors, numerous stress signaling pathways are activated in plants. Among these, the mitogen-activated protein kinase (MAPK) signaling cascade plays a pivotal role in diverse plant stress responses. MPK3 and MPK6 function in several cellular signaling pathways by phosphorylating downstream partner proteins in response to environmental stresses. However, little is known about the MPK3/MPK6 signaling pathway in sweetpotato [Ipomoea batatas (L.) Lam]. We recently confirmed that IbMPK3 and IbMPK6, two pathogen-responsive MAPKs, play essential roles in defense gene activation in sweetpotato. In this study, we show that sweetpotato SP8-binding factor (IbSPF1), a substrate of IbMPK3/IbMPK6, functions as a transcriptional regulator of biotic stress signaling in sweetpotato. IbSPF1 specifically interacts with IbMPK3 and IbMPK6, which phosphorylate Ser75 and Ser110 residues of IbSPF1. This increases the affinity of IbSPF1 for the W-box element in target gene promoters. Additionally, the expression of IbSPF1 was up-regulated under various stress conditions and different hormone treatments involved in plant defense responses. Interestingly, the phospho-mimicking mutant of IbSPF1 showed enhanced resistance to Pseudomonas syringae pv. tabaci, and transient expression of mutant IbSPF1 induced the expression of pathogenesis-related genes. These results indicate that the phosphorylation of IbSPF1 by IbMPK3/IbMPK6 plays a critical role in plant immunity by up-regulating the expression of downstream genes.

Sweetpotato [Ipomoea batatas (L.) Lam], which contains high levels of antioxidants such as ascorbate and carotenoids in its storage root, is one of the healthiest foods, as well as one of the best starch crops for growth on marginal lands. In plants, carotenoid pigments are involved in light harvesting for photosynthesis and are also essential for photo-protection against excess light. As dietary antioxidants in humans, these compounds benefit health by alleviating aging-related diseases. The storage root of sweetpotato is a good source of both carotenoids and carbohydrates for human consumption. Therefore, metabolic engineering of sweetpotato to increase the content of useful carotenoids represents an important agricultural goal. This effort has been facilitated by cloning of most of the carotenoid biosynthetic genes, as well as the Orange gene involved in carotenoid accumulation. In this review, we describe our current understanding of the regulation of biosynthesis, accumulation and catabolism of carotenoids in sweetpotato. A deeper understanding of these topics should contribute to development of new sweetpotato cultivars with higher levels of nutritional carotenoids and abiotic stress tolerance.

Phosphorylation of substrate proteins by mitogen-activated protein kinases (MPKs) determines the specific cellular responses elicited by a particular extracellular stimulus. However, downstream targets of plant MPKs remain poorly characterized. In this study, 29 putative substrates of AtMPK3, AtMPK4 and AtMPK6 were identified by solid-phase phosphorylation screening of a λ phage expression library constructed from combined mRNAs from salt-treated, pathogen-treated and mechanically wounded Arabidopsis seedlings. To test the efficiency of this screening, we performed in vitro kinase assay with 10 recombinant fusion proteins. All proteins were phosphorylated by AtMPK3, AtMPK4 and AtMPK6, indicating the efficiency of this screening procedure. To confirm phosphorylation of isolated substrates by plant MPKs, we performed in-gel kinase assays. All test substrates were strongly phosphorylated by wounding or H2O2-activated AtMPK3 and AtMPK6. Three substrates, encoded by genes At2g41430, At2g41900, and At3g16770, were strongly phosphorylated, suggesting a function as AtMPK substrates. The type of screening provides a powerful way for identifying potential substrates of MAP kinases responsive to biotic and abiotic stresses.

Summary The flowering time regulator GIGANTEA (GI) connects networks involved in developmental stage transitions and environmental stress responses in Arabidopsis. However, little is known about the role of GI in growth, development, and responses to environmental challenges in the perennial plant poplar. Here, we identified and functionally characterized three GI-like genes (PagGIa, PagGIb, and PagGIc) from poplar (Populus alba × P. glandulosa). PagGIs are predominantly nuclear localized and their transcripts are rhythmically expressed, with a peak around zeitgeber time 12 under long-day conditions. Overexpressing PagGIs in wild-type Arabidopsis induced early flowering and salt sensitivity, while overexpressing PagGIs in the gi-2 mutant completely or partially rescued its delayed flowering and enhanced salt tolerance phenotypes. Furthermore, the PagGIs-PagSOS2 complexes inhibited PagSOS2-regulated phosphorylation of PagSOS1 in the absence of stress, whereas these inhibitions were eliminated due to the degradation of PagGIs under salt stress. Down-regulation of PagGIs by RNA interference led to vigorous growth, higher biomass, and enhanced salt stress tolerance in transgenic poplar plants. Taken together, these results indicate that several functions of Arabidopsis GI are conserved in its poplar orthologs, and they lay the foundation for developing new approaches to producing salt-tolerant trees for sustainable development on marginal lands worldwide. This article is protected by copyright. All rights reserved.

Tocopherol (vitamin E) is a chloroplast lipid that is presumed to be involved in the plant response to oxidative stress. In this study, we isolated and characterized five tocopherol biosynthetic genes from sweetpotato (Ipomoea batatas [L.] Lam) plants, including genes encoding 4-hydroxyphenylpyruvate dioxygenase (IbHPPD), homogentisate phytyltransferase (IbHPT), 2-methyl-6-phytylbenzoquinol methyltransferase (IbMPBQ MT), tocopherol cyclase (IbTC) and γ-tocopherol methyltransferase (IbTMT). Fluorescence microscope analysis indicated that four proteins localized into the chloroplast, whereas IbHPPD observed in the nuclear. Quantitative RT-PCR analysis revealed that the expression patterns of the five tocopherol biosynthetic genes varied in different plant tissues and under different stress conditions. All five genes were highly expressed in leaf tissues, whereas IbHPPD and IbHPT were highly expressed in the thick roots. The expression patterns of these five genes significantly differed in response to PEG, NaCl and H2O2-mediated oxidative stress. IbHPPD was strongly induced following PEG and H2O2 treatment and IbHPT was strongly induced following PEG treatment, whereas IbMPBQ MT and IbTC were highly expressed following NaCl treatment. Upon infection of the bacterial pathogen Pectobacterium chrysanthemi, the expression of IbHPPD increased sharply in sweetpotato leaves, whereas the expression of the other genes was reduced or unchanged. Additionally, transient expression of the five tocopherol biosynthetic genes in tobacco (Nicotiana bentamiana) leaves resulted in increased transcript levels of the transgenes expressions and tocopherol production. Therefore, our results suggested that the five tocopherol biosynthetic genes of sweetpotato play roles in the stress defense response as transcriptional regulators of the tocopherol production.

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&ndash, 19.6 and 3.2-fold higher, respectively, than those of non-transgenic (NT) storage roots (white flesh). The &beta, 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 &deg, 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.

Carotenoids are required for many biological processes in plants and humans. Lycopene e-cyclase (LCY-e) catalyzes the conversion of lycopene into lutein via the α-branch carotenoid biosynthesis pathway. Down-regulation of IbLCY-e by RNAi increases carotenoid accumulation and salt stress tolerance in transgenic sweetpotato calli. As the role of IbLCY-e in carotenoid biosynthesis and environmental stress responses in whole plants is poorly understood, transgenic sweetpotato (RLE plants) with reduced expression of IbLCY-e were developed. RLE plants contained higher levels of total carotenoid and β-carotene, due to an elevated β-carotene/lutein ratio rather than increased de novo biosynthesis. RLE plants showed high reactive oxygen species/radical-scavenging activity. They also exhibited an enhanced tolerance of both salt and drought stress, which was associated with lower membrane permeability and a higher photosynthetic rate, respectively. Elevated carotenoid accumulation in RLE plants mitigated the reductions in leaf photosystem II efficiency and chlorophyll induced by abiotic stress. Expression of the carotenoid cleavage genes 9-cis-epoxycarotenoid dioxygenase, carotenoid cleavage dioxygenase 1 (CCD1) and CCD4 was higher in RLE plants, as was abscisic acid accumulation. IbLCY-e silencing thus offers an effective approach for developing sweetpotato plants with increased tolerance to abiotic stress that will grow on global marginal lands with no reduction in nutritional value.

Dehydration-responsive element-binding/C-repeat-binding factor (DREB/CBF) proteins regulate the transcription of genes involved in cold acclimation in several species. However, little is known about the physiological functions of CBF proteins in the low temperature-sensitive crop sweetpotato. We previously reported that the DREB1/CBF-like sweetpotato gene SwDREB1/IbCBF3 is involved in responses to diverse abiotic stresses. In this study, we confirmed that IbCBF3 is localized to the nucleus and binds to the C-repeat/dehydration-responsive elements (CRT/DRE) in the promoters of cold-regulated (COR) genes. We generated transgenic sweetpotato plants overexpressing IbCBF3 under the control of the CaMV 35S promoter (referred to as SC plants) and evaluated their responses to various abiotic stresses. IbCBF3 expression was dramatically induced by cold and drought but much less strongly induced by high salinity and ABA. We further characterized two SC lines (SC3 and SC6) with high levels of IbCBF3 transcript. The SC plants displayed enhanced tolerance to cold, drought, and oxidative stress on the whole-plant level. Under cold stress treatment (4 °C for 48 h), severe wilting and chilling injury were observed in the leaves of wild-type (WT) plants, whereas SC plants were not affected by cold stress. In addition, the COR genes were significantly upregulated in SC plants compared with the WT. The SC plants also showed significantly higher tolerance to drought stress than the WT, which was associated with higher photosynthesis efficiency and lower hydrogen peroxide levels. These results indicate that IbCBF3 is a functional transcription factor involved in the responses to various abiotic stresses in sweetpotato.

The expression of CBF (C-repeat-binding factor) genes is required for freezing tolerance in Arabidopsis thaliana. CBFs are positively regulated by INDUCER OF CBF EXPRESSION1 (ICE1) and negatively regulated by MYB15. These transcription factors directly interact with specific elements in the CBF promoters. Mitogen-activated protein kinase (MAPK/MPK) cascades function upstream to regulate CBFs. However, the mechanism by which MPKs control CBF expression during cold stress signaling remains unknown. This study showed that the activity of MYB15, a transcriptional repressor of cold signaling, is regulated by MPK6-mediated phosphorylation. MYB15 specifically interacts with MPK6, and MPK6 phosphorylates MYB15 on Ser168. MPK6-induced phosphorylation reduced the affinity of MYB15 binding to the CBF3 promoter and mutation of its phosphorylation site (MYB15S168A) enhanced the transcriptional repression of CBF3 by MYB15. Furthermore, transgenic plants overexpressing MYB15S168A showed significantly reduced CBF transcript levels in response to cold stress, compared with plants overexpressing MYB15. The MYB15S168A-overexpressing plants were also more sensitive to freezing than MYB15-overexpressing plants. These results suggest that MPK6-mediated regulation of MYB15 plays an important role in cold stress signaling in Arabidopsis.

β-carotene, a carotenoid that plays a key photo-protective role in plants is converted into zeaxanthin by β-carotene hydroxylase (CHY-β). Previous work showed that down-regulation of IbCHY-β by RNA interference (RNAi) results in higher levels of β-carotene and total carotenoids, as well as salt stress tolerance, in cultured transgenic sweetpotato cells. In this study, we introduced the RNAi-IbCHY-β 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-β transcript levels. The RC plants had orange flesh, total carotenoid and β-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 β-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.

Carotenoids have essential roles in light-harvesting processes and protecting the photosynthetic machinery from photo-oxidative damage. Phytoene synthase (PSY) and Orange (Or) are key plant proteins for carotenoid biosynthesis and accumulation. We previously isolated the sweetpotato (Ipomoea batatas) Or gene (IbOr), which is involved in carotenoid accumulation and salt stress tolerance. The molecular mechanism underlying IbOr regulation of carotenoid accumulation was unknown. Here, we show that IbOr has an essential role in regulating IbPSY stability via its holdase chaperone activity both in vitro and in vivo. This protection results in carotenoid accumulation and abiotic stress tolerance. IbOr transcript levels increase in sweetpotato stem, root, and calli after exposure to heat stress. IbOr is localized in the nucleus and chloroplasts, but interacts with IbPSY only in chloroplasts. After exposure to heat stress, IbOr predominantly localizes in chloroplasts. IbOr overexpression in transgenic sweetpotato and Arabidopsis conferred enhanced tolerance to heat and oxidative stress. These results indicate that IbOr holdase chaperone activity protects IbPSY stability, which leads to carotenoid accumulation, and confers enhanced heat and oxidative stress tolerance in plants. This study provides evidence that IbOr functions as a molecular chaperone, and suggests a novel mechanism regulating carotenoid accumulation and stress tolerance in plants.

Plants are continually exposed to numerous environmental stresses. To decrease damage caused by these potentially detrimental factors, various stress-related signaling cascades are activated in plants. One such stress-responsive signaling pathway, the mitogen-activated protein kinase (MAPK) module, plays a critical role in diverse plant stress responses. Here, we functionally characterized biotic and abiotic stress-responsive MAPK genes, IbMPK3 and IbMPK6, from sweetpotato. IbMPK3/6 contain totally 11 MAPK conserved subdomains and the phosphorylating motif TEY. Bacterially expressed IbMPK3/6 could be autophosphorylated in vitro, and these proteins phosphorylated universal kinase substrate, such as myelin basic protein. IbMPK3/6 transcripts were expressed in leaf, stem, and root of sweetpotato cultivars with storage roots of various colors. IbMPK3 and IbMPK6 were induced by various biotic/abiotic stress treatments. Furthermore, the kinase activity of IbMPK3/6 was induced during early NaCl, SA, H2O2, and ABA treatment. IbMPK3/6 were predominantly localized to the nucleus. To determine the biological functions of IbMPK3/6, we transiently expressed the IbMPK genes in tobacco (Nicotiana benthamiana) leaves, which resulted in enhanced tolerance to bacterial pathogen and increased expression of pathogenesis-related (PR) genes. These data demonstrate that IbMPK3 and IbMPK6 play significant roles in plant responses to environmental stress.

In plant cells, the vacuole functions as a major calcium store. Although a calmodulin-regulated Ca2+-ATPase (ACA4) is known to be present in prevacuolar compartments, the presence of an ACA-type Ca2+-ATPase in the mature vacuole of a plant cell has not been verified. Here we provide evidence that ACA11 localizes to the vacuole membrane. ACA11 tagged with GFP was expressed in stable transgenic plants, and visualized in root cells and protoplasts by confocal microscopy. A Ca2+-ATPase function for ACA11 was confirmed by complementation of yeast mutants. A calmodulin binding domain was identified within the first 37 residues of the N-terminal autoinhibitory region.