Haplotype-based phylogenetic analysis and population genomics uncover the origin and domestication of sweetpotato.

The hexaploid sweetpotato (Ipomoea batatas) is one of the most important root crops worldwide. However, its genetic origin remains controversial, and its domestication history remains unknown. In this study, we used a range of genetic evidence and a newly developed haplotype-based phylogenetic analysis to identify two probable progenitors of sweetpotato. The diploid progenitor was likely closely related to Ipomoea aequatoriensis and contributed the B1 subgenome, IbT-DNA2, and the lineage 1 type of chloroplast genome to sweetpotato. The tetraploid progenitor of sweetpotato was most likely I. batatas 4x, which donated the B2 subgenome, IbT-DNA1, and the lineage 2 type of chloroplast genome. Sweetpotato most likely originated from reciprocal crosses between the diploid and tetraploid progenitors, followed by a subsequent whole-genome duplication. In addition, we detected biased gene exchanges between the subgenomes; the rate of B1 to B2 subgenome conversions was nearly three times higher than that of B2 to B1 subgenome conversions. Our analyses revealed that genes involved in storage root formation, maintenance of genome stability, biotic resistance, sugar transport, and potassium uptake were selected during the speciation and domestication of sweetpotato. This study sheds light on the evolution of sweetpotato and paves the way for improvement of this crop.

Sweet potato ADP-glucose pyrophosphorylase small subunit affects vegetative growth, starch content and storage root yield.

The development of storage roots is a key factor determining the yields of crop plants, including sweet potato. Here, using combined bioinformatic and genomic approaches, we identified a sweet potato yield-related gene, ADP-glucose pyrophosphorylase (AGP) small subunit (IbAPS). We found that IbAPS positively affects AGP activity, transitory starch biosynthesis, leaf development, chlorophyll metabolism, and photosynthesis, ultimately affecting the source strength. IbAPS overexpression in sweet potato led to increased vegetative biomass and storage root yield. RNAi of IbAPS resulted in reduced vegetative biomass, accompanied with a slender stature and stunted root development. In addition to the effects on root starch metabolism, we found that IbAPS affects other storage root development-associated events, including lignification, cell expansion, transcriptional regulation, and production of the storage protein sporamins. A combinatorial analysis based on transcriptomes, as well as morphological and physiological data, revealed that IbAPS affects several pathways that determine development of vegetative tissues and storage roots. Our work establishes an important role of IbAPS in concurrent control of carbohydrate metabolism, plant growth, and storage root yield. We showed that upregulation of IbAPS results in superior sweet potato with increased green biomass, starch content, and storage root yield. The findings expand our understanding of the functions of AGP enzymes and advances our ability to increase the yield of sweet potato and, perhaps, other crop plants.

Modulation of anthocyanin accumulation in storage roots of sweetpotato by transcription factor IbMYB1-2 through direct binding to anthocyanin biosynthetic gene promoters.

The storage roots of purple-fleshed sweetpotato rich in anthocyanins are considered nutrient-rich foods with health effects. However, the molecular mechanism underlying anthocyanin biosynthesis and regulation remains to be revealed. In this study, IbMYB1-2 was isolated from purple-fleshed sweetpotato "Xuzishu8". The phylogenetic and sequence analysis indicated that IbMYB1-2 belongs to the SG6 subfamily with a conserved bHLH motif. Subcellular localization analysis and transcriptional activity assay revealed that IbMYB1-2 is a key transcriptional activator and is specific to the nucleus. Agrobacterium rhizogenes-mediated overexpression of IbMYB1-2 in sweetpotato through in vivo root transgenic system led to an increase in anthocyanins in the root of sweetpotato. qRT-PCR and transcriptome analysis depicted that the transcript levels of IbMYB1-2, IbbHLH42, and eight structural genes that are associated with the synthesis of anthocyanin were upregulated in overexpressed IbMYB1-2 transgenic roots. Dual-luciferase reporter (DLR) assay and yeast one-hybrid (Y1H) assay demonstrated IbMYB1-2 binding to the promoter regions of IbbHLH42 and other anthocyanin biosynthetic genes, including IbCHS, IbCHI, IbF3H, IbDFR, IbANS, IbGSTF12, IbUGT78D2, and IbUF3GT. Moreover, IbbHLH42 was shown to be an active enhancer for the formation of MYB-bHLH-WD40 (MBW) complex, which strongly supports the promoter activities of the IbCHS, IbANS, IbUGT78D2, and IbGSTF12 genes to induce anthocyanin accumulation. Taken together, our findings not only revealed the underlying regulatory molecular mechanism of IbMYB1-2 for anthocyanin accumulation in the storage roots of sweetpotato but also uncovered a potential mechanism by which IbbHLH42 modulated anthocyanin biosynthesis through a positive feedback regulatory loop.

Engineering Properties of Sweet Potato Starch for Industrial Applications by Biotechnological Techniques including Genome Editing.

Sweet potato (Ipomoea batatas) is one of the largest food crops in the world. Due to its abundance of starch, sweet potato is a valuable ingredient in food derivatives, dietary supplements, and industrial raw materials. In addition, due to its ability to adapt to a wide range of harsh climate and soil conditions, sweet potato is a crop that copes well with the environmental stresses caused by climate change. However, due to the complexity of the sweet potato genome and the long breeding cycle, our ability to modify sweet potato starch is limited. In this review, we cover the recent development in sweet potato breeding, understanding of starch properties, and the progress in sweet potato genomics. We describe the applicational values of sweet potato starch in food, industrial products, and biofuel, in addition to the effects of starch properties in different industrial applications. We also explore the possibility of manipulating starch properties through biotechnological means, such as the CRISPR/Cas-based genome editing. The ability to target the genome with precision provides new opportunities for reducing breeding time, increasing yield, and optimizing the starch properties of sweet potatoes.

Dynamic network biomarker analysis discovers IbNAC083 in the initiation and regulation of sweet potato root tuberization.

The initiation and development of storage roots (SRs) are intricately regulated by a transcriptional regulatory network. One key challenge is to accurately pinpoint the tipping point during the transition from pre-swelling to SRs and to identify the core regulators governing such a critical transition. To solve this problem, we performed a dynamic network biomarker (DNB) analysis of transcriptomic dynamics during root development in Ipomoea batatas (sweet potato). First, our analysis identified stage-specific expression patterns for a significant proportion (>9%) of the sweet potato genes and unraveled the chronology of events that happen at the early and later stages of root development. Then, the results showed that different root developmental stages can be depicted by co-expressed modules of sweet potato genes. Moreover, we identified the key components and transcriptional regulatory network that determine root development. Furthermore, through DNB analysis an early stage, with a root diameter of 3.5 mm, was identified as the critical period of SR swelling initiation, which is consistent with morphological and metabolic changes. In particular, we identified a NAM/ATAF/CUC (NAC) domain transcription factor, IbNAC083, as a core regulator of this initiation in the DNB-associated network. Further analyses and experiments showed that IbNAC083, along with its associated differentially expressed genes, induced dysfunction of metabolism processes, including the biosynthesis of lignin, flavonol and starch, thus leading to the transition to swelling roots.

The H+-pyrophosphatase IbVP1 regulates carbon flux to influence the starch metabolism and yield of sweet potato.

Storage roots of sweet potato are important sink organs for photoassimilates and energy, and carbohydrate metabolism in storage roots affects yield and starch production. Our previous study showed that sweet potato H+-pyrophosphatase (IbVP1) plays a vital role in mitigating iron deficiency and positively controls fibrous root growth. However, its roles in regulating starch production in storage roots have not been investigated. In this study, we found that IbVP1 overexpression in sweet potato improved the photosynthesis ability of and sucrose content in source leaves and increased both the starch content in and total yield of sink tissues. Using 13C-labeled sucrose feeding, we determined that IbVP1 overexpression promotes phloem loading and sucrose long-distance transport and enhances Pi-use efficiency. In sweet potato plants overexpressing IbVP1, the expression levels of starch biosynthesis pathway genes, especially AGPase and GBSSI, were upregulated, leading to changes in the structure, composition, and physicochemical properties of stored starch. Our study shows that the IbVP1 gene plays an important role in regulating starch metabolism in sweet potato. Application of the VP1 gene in genetic engineering of sweet potato cultivars may allow the improvement of starch production and yield under stress or nutrient-limited conditions.

Genome-wide identification of agronomically important genes in outcrossing crops using OutcrossSeq.

Many important crops (e.g., tuber, root, and tree crops) are cross-pollinating. For these crops, no inbred lines are available for genetic study and breeding because they are self-incompatible, clonally propagated, or have a long generation time, making the identification of agronomically important genes difficult, particularly in crops with a complex autopolyploid genome. In this study, we developed a method, OutcrossSeq, for mapping agronomically important loci in outcrossing crops based on whole-genome low-coverage resequencing of a large genetic population, and designed three computation algorithms in OutcrossSeq for different types of outcrossing populations. We applied OutcrossSeq to a tuberous root crop (sweet potato, autopolyploid), a tree crop (walnut tree, highly heterozygous diploid), and hybrid crops (double-cross populations) to generate high-density genotype maps for the outcrossing populations, which enable precise identification of genomic loci underlying important agronomic traits. Candidate causative genes at these loci were detected based on functional clues. Taken together, our results indicate that OutcrossSeq is a robust and powerful method for identifying agronomically important genes in heterozygous species, including polyploids, in a cost-efficient way. The OutcrossSeq software and its instruction manual are available for downloading at www.xhhuanglab.cn/tool/OutcrossSeq.html.

Correction to: Improved insect resistance against Spodoptera litura in transgenic sweetpotato by overexpressing Cry1Aa toxin.

In Materials and method section, a sweetpotato variety "Taizhong-6" (China national number 2013003) should be renamed as Ayamurasaki".

CRISPR/Cas9-Based Mutagenesis of Starch Biosynthetic Genes in Sweet Potato (Ipomoea Batatas) for the Improvement of Starch Quality.

CRISPR/Cas9-mediated genome editing is a powerful technology that has been used for the genetic modification of a number of crop species. In order to evaluate the efficacy of CRISPR/Cas9 technology in the root crop, sweet potato (Ipomoea batatas), two starch biosynthetic pathway genes, IbGBSSI (encoding granule-bound starch synthase I), and IbSBEII (encoding starch branching enzyme II), were targeted in the starch-type cultivar Xushu22 and carotenoid-rich cultivar Taizhong6. I. batatas was transformed using a binary vector, in which the Cas9 gene is driven by the Arabidopsis AtUBQ promoter and the guide RNA is controlled by the Arabidopsis AtU6 promoter. A total of 72 Xushu22 and 35 Taizhong6 transgenic lines were generated and analyzed for mutations. The mutation efficiency was 62-92% with multi-allelic mutations in both cultivars. Most of the mutations were nucleotide substitutions that lead to amino acid changes and, less frequently, stop codons. In addition, short nucleotide insertions or deletions were also found in both IbGBSSI and IbSBEII. Furthermore, a 2658 bp deletion was found in one IbSBEII transgenic line. The total starch contents were not significantly changed in IbGBSSI- and IbSBEII-knockout transgenic lines compared to the wild-type control. However, in the allopolyploid sweet potato, the IbGBSSI-knockout reduced, while the IbSBEII-knockout increased, the amylose percentage. Our results demonstrate that CRISPR/Cas9 technology is an effective tool for the improvement of starch qualities in sweet potato and breeding of polyploid root crops.

Improved insect resistance against Spodoptera litura in transgenic sweetpotato by overexpressing Cry1Aa toxin.

KEY MESSAGE: Overexpressing the Cry1Aa gene in sweetpotato significantly reduced pest damage through disrupting the integrity of the midgut of Spodoptera litura larvae for resistance against target Lepidoptera insect pests in sweetpotato. Sweetpotato is susceptible to insect pests and diseases leading to yield losses during pest outbreaks. Lepidoptera insects such as S litura are especially important pests of sweetpotato. The effect of Cry1Aa gene on S. litura was investigated by overexpressing Cry1Aa gene in sweetpotato to relieve symptoms due to pest damage. When transgenic leaves were fed to the larvae of S. litura, the growth of the larvae was reduced, the larval quality decreased, and mortality was increased compared with the larvae that fed on wild-type leaves. Further anatomical analysis revealed that the columnar cells of the midgut epithelium of the BT group were significantly damaged, loosened, or disordered. Furthermore, the integrity of the midgut was destroyed. In addition, when potted seedlings of the wild-type and BT sweetpotato were inoculated with the same number of S. litura larvae, wild-type plants died on the eighth day after infestation, while BT transgenic lines still grew normally. This study showed that transgenic sweetpotato overexpressing Cry1Aa can prevent S. litura infestation, and thus increase the yield of sweetpotato.

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

KEY MESSAGE: 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 18 days. 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.

A novel glycosyltransferase catalyses the transfer of glucose to glucosylated anthocyanins in purple sweet potato.

Glycosylation contributes to the diversity and stability of anthocyanins in plants. The process is catalysed by various glucosyltransferases using different anthocyanidin aglycones and glycosyl donors. In this study, we found that an anthocyanidin 3-O-glucoside-2″-O-glucosyltransferase (3GGT) from purple sweet potato (Ipomoea batatas) catalyses the conversion of anthocyanidin 3-O-glucoside into anthocyanidin 3-O-sophoroside, which is functionally different from the 3GGT ortholog of Arabidopsis. Phylogenetic analysis indicated regioselectivity of 3GGT using uridine-5'-diphosphate (UDP)-xylose or UDP-glucose as the glycosyl is divergent between Convolvulaceae and Arabidopsis. Homology-based protein modeling and site-directed mutagenesis of Ib3GGT and At3GGT suggested that the Thr-138 of Ib3GGT is a key amino acid residue for UDP-glucose recognition and that it plays a major role in sugar-donor selectivity. Wild-type and ugt79b1 mutants (defective in UDP carbohydrate-dependent glycosyltransferases, UGTs) of Arabidopsis plants overexpressing Ib3GGT produced the new component cyanidin 3-O-sophoroside. Moreover, Ib3GGT expression was associated with anthocyanin accumulation in different tissues during I. batatas plant development and was regulated by the transcription factor IbMYB1. Localization assays for Ib3GGT showed that glycosyl extension occurs in the cytosol and not in the endoplasmic reticulum. This study therefore reveals the function of Ib3GGT in glycosyl extension of anthocyanins and demonstrates that Thr-138 is the key amino acid residue for UDP-glucose recognition.

C4 Protein of Sweet Potato Leaf Curl Virus Regulates Brassinosteroid Signaling Pathway through Interaction with AtBIN2 and Affects Male Fertility in Arabidopsis.

Sweepoviruses have been identified globally and cause substantial yield losses and cultivar decline in sweet potato. This study aimed to investigate the interaction between sweepovirus and plant host by analyzing the function of the viral protein C4 of Sweet potato leaf curl virus-Jiangsu (SPLCV-JS), a sweepovirus cloned from diseased sweet potato plants in East China. Ectopic expression of the C4 in Arabidopsis altered plant development drastically with phenotypic changes including leaf curling, seedling twisting, deformation of floral tissues and reduction of pollen fertility, and seed number. Using bimolecular fluorescence complementation analysis, this study demonstrated that the SPLCV-JS C4 protein interacted with brassinosteroid-insensitive 2 (AtBIN2) in the plasma membrane of Nicotiana benthamiana cells. The C4 AtBIN2 interaction was further confirmed by yeast two-hybrid assays. This interaction led to the re-localization of AtBIN2-interacting proteins AtBES1/AtBZR1 into the nucleus which altered the expression of brassinosteroid (BR)-response genes, resulting in the activation of BR-signaling pathway. The interaction of SPLCV-JS C4 and AtBIN2 also led to the down-regulated expression of key genes involved in anther and pollen development, including SPROROCYTELESS/NOZZLE, DEFECTIVE IN TAPEL DEVELOPMENT AND FUNCTION 1, and ABORTED MICROSPORES, which caused abnormal tapetal development, followed by defective exine pattern formation of microspores and pollen release. Consequently, male fertility in the C4 transgenic Arabidopsis was reduced. The present study illustrated how the sweepovirus C4 protein functioned in host cells and affected male fertility by interacting with the key components of BR-signaling pathway.

Haplotype-resolved sweet potato genome traces back its hexaploidization history.

Here we present the 15 pseudochromosomes of sweet potato, Ipomoea batatas, the seventh most important crop in the world and the fourth most significant in China. By using a novel haplotyping method based on genome assembly, we have produced a half haplotype-resolved genome from ~296 Gb of paired-end sequence reads amounting to roughly 67-fold coverage. By phylogenetic tree analysis of homologous chromosomes, it was possible to estimate the time of two recent whole-genome duplication events as occurring about 0.8 and 0.5 million years ago. This half haplotype-resolved hexaploid genome represents the first successful attempt to investigate the complexity of chromosome sequence composition directly in a polyploid genome, using sequencing of the polyploid organism itself rather than any of its simplified proxy relatives. Adaptation and application of our approach should provide higher resolution in future genomic structure investigations, especially for similarly complex genomes.

H+ -pyrophosphatase IbVP1 promotes efficient iron use in sweet potato [Ipomoea batatas (L.) Lam.].

Iron (Fe) deficiency is one of the most common micronutrient deficiencies limiting crop production globally, especially in arid regions because of decreased availability of iron in alkaline soils. Sweet potato [Ipomoea batatas (L.) Lam.] grows well in arid regions and is tolerant to Fe deficiency. Here, we report that the transcription of type I H+ -pyrophosphatase (H+ -PPase) gene IbVP1 in sweet potato plants was strongly induced by Fe deficiency and auxin in hydroponics, improving Fe acquisition via increased rhizosphere acidification and auxin regulation. When overexpressed, transgenic plants show higher pyrophosphate hydrolysis and plasma membrane H+ -ATPase activity compared with the wild type, leading to increased rhizosphere acidification. The IbVP1-overexpressing plants showed better growth, including enlarged root systems, under Fe-sufficient or Fe-deficient conditions. Increased ferric precipitation and ferric chelate reductase activity in the roots of transgenic lines indicate improved iron uptake, which is also confirmed by increased Fe content and up-regulation of Fe uptake genes, e.g. FRO2, IRT1 and FIT. Carbohydrate metabolism is significantly affected in the transgenic lines, showing increased sugar and starch content associated with the increased expression of AGPase and SUT1 genes and the decrease in ß-amylase gene expression. Improved antioxidant capacities were also detected in the transgenic plants, which showed reduced H2 O2 accumulation associated with up-regulated ROS-scavenging activity. Therefore, H+ -PPase plays a key role in the response to Fe deficiency by sweet potato and effectively improves the Fe acquisition by overexpressing IbVP1 in crops cultivated in micronutrient-deficient soils.

Altered Phenylpropanoid Metabolism in the Maize Lc-Expressed Sweet Potato (Ipomoea batatas) Affects Storage Root Development.

There is no direct evidence of the effect of lignin metabolism on early storage root development in sweet potato. In this study, we found that heterologous expression of the maize leaf color (Lc) gene in sweet potato increased anthocyanin pigment accumulation in the whole plant and resulted in reduced size with an increased length/width ratio, low yield and less starch content in the early storage roots. RT-PCR analysis revealed dramatic up-regulation of the genes involved in the lignin biosynthesis pathway in developing storage roots, leading to greater lignin content in the Lc transgenic lines, compared to the wild type. This was also evidenced by the enhanced lignification of vascular cells in the early storage roots. Furthermore, increased expression of the ß-amylase gene in leaves and storage roots also accelerated starch degradation and increased the sugar use efficiency, providing more energy and carbohydrate sources for lignin biosynthesis in the Lc transgenic sweet potato. Lesser starch accumulation was observed in the developing storage roots at the initiation stage in the Lc plants. Our study provides experimental evidence of the basic carbohydrate metabolism underlying the development of storage roots, which is the transformation of lignin biosynthesis to starch biosynthesis.

Resistance to Ditylenchus destructor Infection in Sweet Potato by the Expression of Small Interfering RNAs Targeting unc-15, a Movement-Related Gene.

Stem nematode (Ditylenchus destructor) is one of most serious diseases that limit the productivity and quality of sweet potato (Ipomoea batatas), a root crop with worldwide importance for food security and nutrition improvement. Hence, there is a global demand for developing sweet potato varieties that are resistant to the disease. In this study, we have investigated the interference of stem nematode infectivity by the expression of small interfering RNAs (siRNAs) in transgenic sweet potato that are homologous to the unc-15 gene, which affects the muscle protein paramyosin of the pathogen. The production of double-stranded RNAs and siRNAs in transgenic lines with a single transgene integration event was verified by Northern blot analysis. The expression of unc-15 was reduced dramatically in stem nematodes collected from the inoculated storage roots of transgenic plants, and the infection areas of their storage roots were dramatically smaller than that of wild-type (WT). Compared with the WT, the transgenic plants showed increased yield in the stem nematode-infested field. Our results demonstrate that the expression of siRNAs targeting the unc-15 gene of D. destructor is an effective approach in improving stem nematode resistance in sweet potato, in adjunct with the global integrated pest management programs.

Elevated compartmentalization of Na+ into vacuoles improves salt and cold stress tolerance in sweet potato (Ipomoea batatas).

Salinity and low temperature are the main limiting factors for sweet potato (Ipomoea batatas) growth and agricultural productivity. Various studies have shown that plant NHX-type antiporter plays a crucial role in regulating plant tolerance to salt stress by intracellular Na(+) compartmentalization. The Arabidopsis thaliana AtNHX1 gene that encodes a vacuolar Na(+) /H(+) antiporter was introduced into the sweet potato cultivar Xushu-22 by Agrobacterium-mediated transformation to confer abiotic stress tolerance. Stable insertion of AtNHX1 into the sweet potato genome and its expression was confirmed by Southern blot and reverse transcription-polymerase chain reaction (RT-PCR). A remarkably higher Na(+) /H(+) exchange activity of tonoplast membrane from transgenic sweet potato lines (NOE) in comparison with wild-type (WT) plants confirmed the vacuolar antiporter function in mediating Na(+) /H(+) exchange. Under salt stress, NOE plants accumulated higher Na(+) and K(+) levels in their tissues compared with WT plants, maintaining high K(+) /Na(+) ratios. Consequently, NOE plants showed enhanced protection against cell damage due to the increased proline accumulation, preserved cell membrane integrity, enhanced reactive oxygen species (ROS) scavenging (e.g. increased superoxide dismutase activity), and reduced H2 O2 and malondialdehyde (MDA) production. Moreover, the transgenic plants showed improved cold tolerance through multiple mechanisms of action, revealing the first molecular evidence for NHX1 function in cold response. The transgenic plants showed better biomass production and root yield under stressful conditions. These findings demonstrate that overexpressing AtNHX1 in sweet potato renders the crop tolerant to both salt and cold stresses, providing a greater capacity for the use of AtNHX1 in improving crop performance under combined abiotic stress conditions.

Functional characterization of Dihydroflavonol-4-reductase in anthocyanin biosynthesis of purple sweet potato underlies the direct evidence of anthocyanins function against abiotic stresses.

Dihydroflavonol-4-reductase (DFR) is a key enzyme in the catalysis of the stereospecific reduction of dihydroflavonols to leucoanthocyanidins in anthocyanin biosynthesis. In the purple sweet potato (Ipomoea batatas Lam.) cv. Ayamurasaki, expression of the IbDFR gene was strongly associated with anthocyanin accumulation in leaves, stems and roots. Overexpression of the IbDFR in Arabidopsis tt3 mutants fully complemented the pigmentation phenotype of the seed coat, cotyledon and hypocotyl. Downregulation of IbDFR expression in transgenic sweet potato (DFRi) using an RNAi approach dramatically reduced anthocyanin accumulation in young leaves, stems and storage roots. In contrast, the increase of flavonols quercetin-3-O-hexose-hexoside and quercetin-3-O-glucoside in the leaves and roots of DFRi plants is significant. Therefore, the metabolic pathway channeled greater flavonol influx in the DFRi plants when their anthocyanin and proanthocyanidin accumulation were decreased. These plants also displayed reduced antioxidant capacity compared to the wild type. After 24 h of cold treatment and 2 h recovery, the wild-type plants were almost fully restored to the initial phenotype compared to the slower recovery of DFRi plants, in which the levels of electrolyte leakage and hydrogen peroxide accumulation were dramatically increased. These results provide direct evidence of anthocyanins function in the protection against oxidative stress in the sweet potato. The molecular characterization of the IbDFR gene in the sweet potato not only confirms its important roles in flavonoid metabolism but also supports the protective function of anthocyanins of enhanced scavenging of reactive oxygen radicals in plants under stressful conditions.

Improved tolerance to various abiotic stresses in transgenic sweet potato (Ipomoea batatas) expressing spinach betaine aldehyde dehydrogenase.

Abiotic stresses are critical delimiters for the increased productivity and cultivation expansion of sweet potato (Ipomoea batatas), a root crop with worldwide importance. The increased production of glycine betaine (GB) improves plant tolerance to various abiotic stresses without strong phenotypic changes, providing a feasible approach to improve stable yield production under unfavorable conditions. The gene encoding betaine aldehyde dehydrogenase (BADH) is involved in the biosynthesis of GB in plants, and the accumulation of GB by the heterologous overexpression of BADH improves abiotic stress tolerance in plants. This study is to improve sweet potato, a GB accumulator, resistant to multiple abiotic stresses by promoted GB biosynthesis. A chloroplastic BADH gene from Spinacia oleracea (SoBADH) was introduced into the sweet potato cultivar Sushu-2 via Agrobacterium-mediated transformation. The overexpression of SoBADH in the transgenic sweet potato improved tolerance to various abiotic stresses, including salt, oxidative stress, and low temperature. The increased BADH activity and GB accumulation in the transgenic plant lines under normal and multiple environmental stresses resulted in increased protection against cell damage through the maintenance of cell membrane integrity, stronger photosynthetic activity, reduced reactive oxygen species (ROS) production, and induction or activation of ROS scavenging by the increased activity of free radical-scavenging enzymes. The increased proline accumulation and systemic upregulation of many ROS-scavenging genes in stress-treated transgenic plants also indicated that GB accumulation might stimulate the ROS-scavenging system and proline biosynthesis via an integrative mechanism. This study demonstrates that the enhancement of GB biosynthesis in sweet potato is an effective and feasible approach to improve its tolerance to multiple abiotic stresses without causing phenotypic defects. This strategy for trait improvement in sweet potato not only stabilizes yield production in normal soils in unpredictable climates but also provides a novel germplasm for sweet potato production on marginal lands.