Transgenic Sweetpotato Plants Research Articles

Sweet potato NAC transcription factor NAC43 negatively regulates plant growth by causing leaf curling and reducing photosynthetic efficiency.

Leaves comprise one of the most important organs for plant growth and development. Although there have been some reports on leaf development and the establishment of leaf polarity, their regulatory mechanisms are not very clear. In this study, we isolated a NAC (NAM, ATAF, and CUC) transcription factor (TF), i.e., IbNAC43, from Ipomoea trifida, which is a wild ancestor of sweet potato. This TF was highly expressed in the leaves and encoded a nuclear localization protein. The overexpression of IbNAC43 caused leaf curling and inhibited the growth and development of transgenic sweet potato plants. The chlorophyll content and photosynthetic rate in transgenic sweet potato plants were significantly lower than those in wild-type (WT) plants. Scanning electron microscopy (SEM) and paraffin sections showed that the ratio of cells in the upper and lower epidermis of the transgenic plant leaves was unbalanced; moreover, the abaxial epidermal cells were irregular and uneven in transgenic plants. In addition, the xylem of transgenic plants was more developed than that of WT plants, while their lignin and cellulose contents were significantly higher than those of WT. Quantitative real-time PCR (qRT-PCR) analysis showed that the overexpression of IbNAC43 upregulated the genes involved in leaf polarity development and lignin biosynthesis in transgenic plants. Moreover, it was found that IbNAC43 could directly activate the expression of the leaf adaxial polarity-related genes IbREV and IbAS1 by binding to their promoters. These results indicate that IbNAC43 might play a critical role in plant growth by affecting the establishment of leaf adaxial polarity. This study provides new insights regarding leaf development.

The transcription factor IbNAC29 positively regulates the carotenoid accumulation in sweet potato.

Carotenoid is a tetraterpene pigment beneficial for human health. Although the carotenoid biosynthesis pathway has been extensively studied in plants, relatively little is known about their regulation in sweet potato. Previously, we conducted the transcriptome database of differentially expressed genes between the sweet potato (Ipomoea batatas) cultivar 'Weiduoli' and its high-carotenoid mutant 'HVB-3'. In this study, we selected one of these candidate genes, IbNAC29, for subsequent analyses. IbNAC29 belongs to the plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factor family. Relative IbNAC29 mRNA level in the HVB-3 storage roots was ~1.71-fold higher than Weiduoli. Additional experiments showed that the contents of α-carotene, lutein, β-carotene, zeaxanthin, and capsanthin are obviously increased in the storage roots of transgenic sweet potato plants overexpressing IbNAC29. Moreover, the levels of carotenoid biosynthesis genes in transgenic plants were also up-regulated. Nevertheless, yeast one-hybrid assays indicated that IbNAC29 could not directly bind to the promoters of these carotenoid biosynthesis genes. Furthermore, the level of IbSGR1 was down-regulated, whose homologous genes in tomato can negatively regulate carotene accumulation. Yeast three-hybrid analysis revealed that the IbNAC29-IbMYB1R1-IbAITR5 could form a regulatory module. Yeast one-hybrid, electrophoretic mobility shift assay, quantitative PCR analysis of chromatin immunoprecipitation and dual-luciferase reporter assay showed that IbAITR5 directly binds to and inhibits the promoter activity of IbSGR1, up-regulating carotenoid biosynthesis gene IbPSY. Taken together, IbNAC29 is a potential candidate gene for the genetic improvement of nutritive value in sweet potato.

Comparative Transcriptome and Interaction Protein Analysis Reveals the Mechanism of IbMPK3-Overexpressing Transgenic Sweet Potato Response to Low-Temperature Stress.

The sweet potato is very sensitive to low temperature. Our previous study revealed that IbMPK3-overexpressing transgenic sweet potato (M3) plants showed stronger low-temperature stress tolerance than wild-type plants (WT). However, the mechanism of M3 plants in response to low-temperature stress is unclear. To further analyze how IbMPK3 mediates low-temperature stress in sweet potato, WT and M3 plants were exposed to low-temperature stress for 2 h and 12 h for RNA-seq analysis, whereas normal conditions were used as a control (CK). In total, 3436 and 8718 differentially expressed genes (DEGs) were identified in WT at 2 h (vs. CK) and 12 h (vs. CK) under low-temperature stress, respectively, whereas 1450 and 9291 DEGs were detected in M3 plants, respectively. Many common and unique DEGs were analyzed in WT and M3 plants. DEGs related to low temperature were involved in Ca2+ signaling, MAPK cascades, the reactive oxygen species (ROS) signaling pathway, hormone transduction pathway, encoding transcription factor families (bHLH, NAC, and WRKY), and downstream stress-related genes. Additionally, more upregulated genes were associated with the MAPK pathway in M3 plants during short-term low-temperature stress (CK vs. 2 h), and more upregulated genes were involved in secondary metabolic synthesis in M3 plants than in the WT during the long-time low-temperature stress treatment (CK vs. 12 h), such as fatty acid biosynthesis and elongation, glutathione metabolism, flavonoid biosynthesis, carotenoid biosynthesis, and zeatin biosynthesis. Moreover, the interaction proteins of IbMPK3 related to photosynthesis, or encoding CaM, NAC, and ribosomal proteins, were identified using yeast two-hybrid (Y2H). This study may provide a valuable resource for elucidating the sweet potato low-temperature stress resistance mechanism, as well as data to support molecular-assisted breeding with the IbMPK3 gene.

A Novel WRKY Transcription Factor from Ipomoea trifida, ItfWRKY70, Confers Drought Tolerance in Sweet Potato.

WRKY transcription factors are one of the important families in plants, and have important roles in plant growth, abiotic stress responses, and defense regulation. In this study, we isolated a WRKY gene, ItfWRKY70, from the wild relative of sweet potato Ipomoea trifida (H.B.K.) G. Don. This gene was highly expressed in leaf tissue and strongly induced by 20% PEG6000 and 100 μM abscisic acid (ABA). Subcellar localization analyses indicated that ItfWRKY70 was localized in the nucleus. Overexpression of ItfWRKY70 significantly increased drought tolerance in transgenic sweet potato plants. The content of ABA and proline, and the activity of SOD and POD were significantly increased, whereas the content of malondialdehyde (MDA) and H2O2 were decreased in transgenic plants under drought stress. Overexpression of ItfWRKY70 up-regulated the genes involved in ABA biosynthesis, stress-response, ROS-scavenging system, and stomatal aperture in transgenic plants under drought stress. Taken together, these results demonstrated that ItfWRKY70 plays a positive role in drought tolerance by accumulating the content of ABA, regulating stomatal aperture and activating the ROS scavenging system in sweet potato.

Inhibition of miR397 by STTM technology to increase sweetpotato resistance to SPVD

As a critical food crop, sweetpotato (Ipomoea batatas (L.) Lam.) is widely planted all over the world, but it is deeply affected by Sweetpotato Virus Disease (SPVD). The present study utilized short tandem target mimic (STTM) technology to effectively up-regulate the expression of laccase (IbLACs) by successfully inhibiting the expression of miR397. The upstream genes in the lignin synthesis pathway were widely up-regulated by feedback regulation, including phenylalanine ammonialyase (PAL), 4-coumarate-CoAligase (4CL), hydroxycinnamoyl CoA:shikimatetransferase (HTC), caffeicacid O-methyltransferase (COMT), and cinnamyl alcohol dehydrogenase (CAD). Meanwhile, the activities of PAL and LAC increased significantly, finally leading to increased lignin content. Lignin deposition in the cell wall increased the physical defence ability of transgenic sweetpotato plants, reduced the accumulation of SPVD transmitted by Bemisia tabaci (Gennadius), and promoted healthy sweetpotato growth. The results provide new insights for disease resistance breeding and green production of sweetpotato.

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.

Targeting of SPCSV-RNase3 via CRISPR-Cas13 confers resistance against sweet potato virus disease.

Sweet potato (Ipomoea batatas) is one of the most important crops in the world, and its production rate is mainly decreased by the sweet potato virus disease (SPVD) caused by the co‐infection of sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus. However, methods for improving SPVD resistance have not been established. Thus, this study aimed to enhance SPVD resistance by targeting one of its important pathogenesis‐related factors (i.e., SPCSV‐RNase3) by using the CRISPR‐Cas13 technique. First, the RNA targeting activity of four CRISPR‐Cas13 variants were compared using a transient expression system in Nicotiana benthamiana. LwaCas13a and RfxCas13d had more efficient RNA and RNA virus targeting activity than PspCas13b and LshCas13a. Driven by the pCmYLCV promoter for the expression of gRNAs, RfxCas13d exhibited higher RNA targeting activity than that driven by the pAtU6 promoter. Furthermore, the targeting of SPCSV‐RNase3 using the LwaCas13a system inhibited its RNA silencing suppressor activity and recovered the RNA silencing activity in N. benthamiana leaf cells. Compared with the wild type, transgenic N. benthamiana plants carrying an RNase3‐targeted LwaCas13a system exhibited enhanced resistance against turnip mosaic virus TuMV‐GFP and cucumber mosaic virus CMV‐RNase3 co‐infection. Moreover, transgenic sweet potato plants carrying an RNase3‐targeted RfxCas13d system exhibited substantially improved SPVD resistance. This method may contribute to the development of SPVD immune germplasm and the enhancement of sweet potato production in SPVD‐prevalent regions.

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.

Downregulation of swpa4 peroxidase expression in transgenic sweetpotato plants decreases abiotic stress tolerance and reduces stress-related peroxidase expression

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.

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

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

Carotenoid cleavage dioxygenase 4 (CCD4) cleaves β-carotene and interacts with IbOr in sweetpotato

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.

A sucrose non-fermenting-1-related protein kinase-1 gene, IbSnRK1, confers salt, drought and cold tolerance in sweet potato

Sucrose non-fermenting-1-related protein kinase-1 (SnRK1) regulates carbon and nitrogen metabolism in plants. However, its roles and their underlying mechanisms in tolerance to abiotic stresses are little known. The present study indicated that the IbSnRK1 gene was strongly induced by NaCl, polyethylene glycol (PEG) 6000, hydrogen peroxide (H2O2), cold (4 °C), and abscisic acid (ABA). Its overexpression significantly increased salt, drought, and cold tolerance in transgenic sweet potato plants. ABA, proline, and K+ contents were significantly increased, whereas malondialdehyde (MDA), Na+ and H2O2 contents and O2− production rate were significantly decreased in the transgenic plants under salt, drought, and cold stresses. Overexpression of the gene up-regulated genes involved in ABA biosynthesis, stress response, and stomatal closure; increased enzyme activities in the reactive oxygen species (ROS) scavenging system; and controlled stomatal closure under salt, drought, and cold stresses. These results show that the IbSnRK1 gene confers salt, drought, and cold tolerance in sweet potato by activating the ROS scavenging system and controlling stomatal closure via the ABA signaling pathway.

Metabolic engineering of low-molecular-weight antioxidants in sweetpotato

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.

Overexpression of swpa4 peroxidase enhances tolerance to hydrogen peroxide and high salinity-mediated oxidative stress in transgenic sweetpotato plants

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.

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.

NHX1 and eIF4A1-stacked transgenic sweetpotato shows enhanced tolerance to drought stress.

Co-expression of Na+/H+ antiporter NHX1 and DEAD-box RNA helicase eIF4A1 from Arabidopsis positively regulates drought stress tolerance by improving ROS scavenging capacity and maintaining membrane integrity in sweetpotato. Plants evolve multiple strategies for stress adaptation in nature. To improve sweetpotato resistance to drought stress, transgenic sweetpotato plants overexpressing the Arabidopsis Na+/H+ antiporter, NHX1, and the translation initiation factor elF4A1 were characterized for phenotypic traits and physiological performance. Without drought treatment, the NHX1-elF4A1 stacked lines (NE lines) showed normal, vigorous growth comparable to the WT plants. The NE plants showed dense green foliage with delayed leaf senescence and developed more roots than WT plants under drought treatment for 18days. Compared to WT plants, higher level of reactive oxygen scavenging capacity was detected in NE lines as indicated by reduced H2O2 accumulation as well as increased superoxide dismutase activity and proline content. The relative ion leakage and malondialdehyde content were reduced in NE plants, indicating improved maintenance of intact membranes system. Both NE plants and NHX1-overexpressing plants (N lines) showed larger aerial parts and well-developed root system compared to WT plants under the drought stress conditions, likely due to the improved antioxidant capacity. The NE plants showed better ROS scavenging than N-line plants. All N- and NE-line plants produced normal storage roots with similar yields as WT in the field under normal growth conditions. These results demonstrated the potential to enhance sweetpotato productivity through stacking genes that are involved in ion compartmentalization and translation initiation.

Sweetpotato bZIP Transcription Factor IbABF4 Confers Tolerance to Multiple Abiotic Stresses.

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.

Overexpression of Arabidopsis YUCCA6 enhances environment stress tolerance and inhibits storage root formation in sweetpotato

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.

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 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-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.