Weijuan Fan

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.

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.

Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato (Ipomoea batatas [L.] Lam.).

Efficient Agrobacterium tumefaciens-mediated transformation was developed using embryogenic suspension cell cultures of elite sweet potato (Ipomoea batatas [L.] Lam.) cultivars, including Ayamurasaki, Sushu2, Sushu9, Sushu11, Wanshu1, Xushu18 and Xushu22. Embryogenic suspension cultures were established in LCP medium using embryogenic calli induced from apical or axillary buds on an induction medium containing 2 mg l(-1) 2,4-D. Suspension cultures were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with the hpt gene as a selectable marker and an intron-interrupted uidA gene as a visible marker. Several key steps of the sweet potato transformation system have been investigated and optimized, including the appropriate antibiotics and their concentrations for suppressing Agrobacterium growth and the optimal doses of hygromycin for transformant selection. A total of 485 putative transgenic plant lines were produced from the transformed calli via somatic embryogenesis and germination to plants under 10 mg l(-1) hygromycin and 200 mg l(-1) cefotaxime. PCR, GUS and Southern blot analyses of the regenerated plants showed that 92.35% of them were transgenic. The number of T-DNA insertions varied from one to three in most transgenic plant lines. Plants showed 100% survival when 308 transgenics were transferred to soil in the greenhouse and then to the field. Most of them were morphologically normal, with the production of storage roots after 3 months of cultivation in the greenhouse or fields. The development of such a robust transformation method suitable to a range of sweet potato genotypes not only provides a routine tool for genetic improvement via transgenesis but also allows us to conduct a functional verification of endogenous genes in sweet potato.

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.Assembly of polyploid plant genomes has been technically challenging. Now, a study presents a half haplotype-resolved hexaploid genome of sweet potato, Ipomoea batatas, using a novel haplotyping method.

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.

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 β-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.

The haplotype-resolved genome sequence of hexaploid Ipomoea batatas reveals its evolutionary history

Although the sweet potato, Ipomoea batatas, is the seventh most important crop in the world and the fourth most significant in China, its genome has not yet been sequenced. The reason, at least in part, is that the genome has proven very difficult to assemble, being hexaploid and highly polymorphic; it has a presumptive composition of two B1 and four B2 component genomes (B1B1B2B2B2B2). By using a novel haplotyping method based on de novo genome assembly, however, we have produced a half haplotype-resolved genome from ∼267Gb of paired-end sequence reads amounting to roughly 60-fold coverage. By phylogenetic tree analysis of homologous chromosomes, it was possible to estimate the time of two whole genome duplication events as occurring about 525,000 and 341,000 years ago. Our analysis also identified many clusters of genes for specialized compounds biosynthesis in this genome. 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 direct sequencing of the polyploid organism itself rather than of 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.]

Summary 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 β‐amylase gene expression. Improved antioxidant capacities were also detected in the transgenic plants, which showed reduced H2O2 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.

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.

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.