Comparative transcriptome profiling of sweetpotato storage roots during curing-mediated wound healing.

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.

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

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.

Overexpression of IbLfp in sweetpotato enhances the low-temperature storage ability of tuberous roots.

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.

Overexpression of 4-hydroxyphenylpyruvate dioxygenase (IbHPPD) increases abiotic stress tolerance in transgenic sweetpotato plants.

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.

Tuberous roots of transgenic sweetpotato overexpressing IbCAD1 have enhanced low-temperature storage phenotypes.

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.

IbINH positively regulates drought stress tolerance in sweetpotato.

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.

Comparative transcriptome profiling of tuberous roots of two sweetpotato lines with contrasting low temperature tolerance during storage.

Sweetpotato (Ipomoea batatas [L.] Lam) is considered an economically important crop worldwide and is used as a source of food, feed, and biomaterials. However, its origin in tropical regions makes it vulnerable to chilling injury during postharvest storage at low temperature. To gain further insight into the molecular mechanism of chilling response, we performed comparative transcriptome analysis of two sweetpotato lines, Xushu 15-1 and Xushu 15-4, with high and low cold storage ability, respectively. Tuberous roots of these lines were stored at 4 °C for 0, 2, and 6 weeks. RNA-Seq data of both lines were de novo assembled, producing 27,636 unigenes with a N50 value of 1204 bp. A total of 525 differentially expressed genes (DEGs) were identified and categorized into six clusters. Genes with higher expression in Xushu 15-1 than in Xushu 15-4 significantly increased in number over time during low temperature storage. Functional annotation of DEGs using KEGG enrichment analysis showed that these DEGs were involved in carbohydrate metabolism, ribosome, protein processing in endoplasmic reticulum, plant-pathogen interaction, and plant hormone signal transduction. Several key candidate genes involved in KEGG pathways were selected and discussed further. The results of this study enhance our understanding of the complex mechanisms involved in low temperature tolerance in sweetpotato during storage and provide a set of candidate genes for the development of new varieties with improved cold storage ability.

Transgenic sweetpotato plants overexpressing tocopherol cyclase display enhanced α-tocopherol content and abiotic stress tolerance.

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.

De novo transcriptome sequencing and gene expression profiling of sweet potato leaves during low temperature stress and recovery.

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.

Down-regulation of lycopene ε-cyclase expression in transgenic sweetpotato plants increases the carotenoid content and tolerance to abiotic stress.

Carotenoids are required for many biological processes in plants and humans. Lycopene ε-cyclase (LCY-ε) catalyzes the conversion of lycopene into lutein via the α-branch carotenoid biosynthesis pathway. Down-regulation of IbLCY-ε by RNAi increases carotenoid accumulation and salt stress tolerance in transgenic sweetpotato calli. As the role of IbLCY-ε in carotenoid biosynthesis and environmental stress responses in whole plants is poorly understood, transgenic sweetpotato (RLE plants) with reduced expression of IbLCY-ε were developed. RLE plants contained higher levels of total carotenoid and ß-carotene, due to an elevated ß-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-ε 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.

Orange: a target gene for regulating carotenoid homeostasis and increasing plant tolerance to environmental stress in marginal lands.

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.

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.

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.

Overexpressing IbCBF3 increases low temperature and drought stress tolerance in transgenic sweetpotato.

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.

Metabolic engineering of carotenoids in transgenic sweetpotato.

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.

Transcriptome profiling of sweetpotato tuberous roots during low temperature storage.

Sweetpotato [Ipomoea batatas (L.) Lam] is a globally important root crop with high industrial value. However, because sweetpotato tuberous roots undergo chilling injuries that negatively affect their quality at temperatures below 10 °C, postharvest damage during the winter season is a major constraint for industrialization. To understand chilling injury response during postharvest low temperature storage, we used next-generation sequencing technology to comprehensive analyze the transcriptome of tuberous roots stored at optimal (13 °C) or low temperature (4 °C) for 6 weeks. From nine cDNA libraries, we produced 298,765,564 clean reads, which were de novo assembled into 58,392 unigenes with an average length of 1100 bp. A total of 3216 differentially expressed genes (DEGs) were detected and categorized into six clusters, of which clusters 2, 4, and 5 (1464 DEGs) were up-regulated under low temperature. The genes in these three clusters are involved in biosynthesis of unsaturated fatty acids, pathogen defense, and phenylalanine metabolism. By contrast, genes in clusters 1, 3, and 6 (1752 DEGs), which were generally down-regulated at low temperature, encode antioxidant enzymes or are involved in glycerophospholipid, carbohydrate, or energy metabolism. We confirmed the results of the transcriptome analysis by quantitative RT-PCR. Our transcriptome analysis will advance our understanding of the comprehensive mechanisms of chilling injury during low temperature storage and facilitate improvements in postharvest storage of sweetpotato tuberous roots.

Down-regulation of GIGANTEA-like genes increases plant growth and salt stress tolerance in poplar.

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 × Populus 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 (WT) 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 orthologues, and they lay the foundation for developing new approaches to producing salt-tolerant trees for sustainable development on marginal lands worldwide.

Orange protein has a role in phytoene synthase stabilization in sweetpotato.

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.

Molecular characterization of biotic and abiotic stress-responsive MAP kinase genes, IbMPK3 and IbMPK6, in sweetpotato.

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.

Molecular characterization of tocopherol biosynthetic genes in sweetpotato that respond to stress and activate the tocopherol production in tobacco.

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.