Transcriptome analysis of sweet potato responses to potassium deficiency.

BACKGROUND: As one of three essential nutrients, potassium is regarded as a main limiting factor for growth and development in plant. Sweet potato (Ipomoea batatas L.) is one of seven major food crops grown worldwide, and is both a nutrient-rich food and a bioenergy crop. It is a typical 'K-favoring' crop, and the level of potassium ion (K+) supplementation directly influences its production. However, little is known about the transcriptional changes in sweet potato genes under low-K+ conditions. Here, we analyzed the transcriptomic profiles of sweet potato roots in response to K+ deficiency to determine the effect of low-K+ stress on this economically important crop. RESULTS: The roots of sweet potato seedlings with or without K+ treatment were harvested and used for transcriptome analyses. The results showed 559 differently expressed genes (DEGs) in low and high K+ groups. Among the DEGs, 336 were upregulated and 223 were downregulated. These DEGs were involved in transcriptional regulation, calcium binding, redox-signaling, biosynthesis, transport, and metabolic process. Further analysis revealed previously unknow genes involved in low-K+ stress, which could be investigated further to improve low K+ tolerance in plants. Confirmation of RNA-sequencing results using qRT-PCR displayed a high level of consistency between the two experiments. Analysis showed that many auxin-, ethylene- and jasmonic acid-related genes respond to K+ deficiency, suggesting that these hormones have important roles in K+ nutrient signaling in sweet potato. CONCLUSIONS: According to the transcriptome data of sweet potato, various DEGs showed transcriptional changes in response to low-K+ stress. However, the expression level of some kinases, transporters, transcription factors (TFs), hormone-related genes, and plant defense-related genes changed significantly, suggesting that they have important roles during K+ deficiency. Thus, this study identifies potential genes for genetic improvement of responses to low-K+ stress and provides valuable insight into the molecular mechanisms regulating low K+ tolerance in sweet potato. Further research is required to clarify the function of these DEGs under low-K+ stress.

Comparative full-length transcriptome analysis by Oxford Nanopore Technologies reveals genes involved in anthocyanin accumulation in storage roots of sweet potatoes (Ipomoea batatas L.).

Background: Storage roots of sweet potatoes (Ipomoea batatas L.) with different colors vary in anthocyanin content, indicating different economically agronomic trait. As the newest DNA/RNA sequencing technology, Oxford Nanopore Technologies (ONT) have been applied in rapid transcriptome sequencing for investigation of genes related to nutrient metabolism. At present, few reports concern full-length transcriptome analysis based on ONT for study on the molecular mechanism of anthocyanin accumulation leading to color change of tuberous roots of sweet potato cultivars. Results: The storage roots of purple-fleshed sweet potato (PFSP) and white-fleshed sweet potato (WFSP) at different developmental stages were subjected to anthocyanin content comparison by UV-visible spectroscopy as well as transcriptome analysis at ONT MinION platform. UV-visible spectrophotometric measurements demonstrated the anthocyanin content of PFSP was much higher than that of WFSP. ONT RNA-Seq results showed each sample generated average 2.75 GB clean data with Full-Length Percentage (FL%) over 70% and the length of N50 ranged from 1,192 to 1,395 bp, indicating reliable data for transcriptome analysis. Subsequent analysis illustrated intron retention was the most prominent splicing event present in the resulting transcripts. As compared PFSP with WFSP at the relative developmental stages with the highest (PH vs. WH) and the lowest (PL vs. WL) anthocyanin content, 282 and 216 genes were up-regulated and two and 11 genes were down-regulated respectively. The differential expression genes involved in flavonoid biosynthesis pathway include CCoAOMT, PpLDOX, DFR, Cytochrome P450, CHI, and CHS. The genes encoding oxygenase superfamily were significantly up-regulated when compared PFSP with WFSP at the relative developmental stages. Conclusions: Comparative full-length transcriptome analysis based on ONT serves as an effective approach to detect the differences in anthocyanin accumulation in the storage roots of different sweet potato cultivars at transcript level, with noting that some key genes can now be closely related to flavonoids biosynthesis. This study helps to improve understanding of molecular mechanism for anthocyanin accumulation in sweet potatoes and also provides a theoretical basis for high-quality sweet potato breeding.

Anthocyanin-Rich Vegetables for Human Consumption-Focus on Potato, Sweetpotato and Tomato.

Malnutrition, unhealthy diets, and lifestyle changes have become major risk factors for non-communicable diseases while adversely impacting economic growth and sustainable development. Anthocyanins, a group of flavonoids that are rich in fruits and vegetables, contribute positively to human health. This review focuses on genetic variation harnessed through crossbreeding and biotechnology-led approaches for developing anthocyanins-rich fruit and vegetable crops. Significant progress has been made in identifying genes involved in anthocyanin biosynthesis in various crops. Thus, the use of genetics has led to the development and release of anthocyanin-rich potato and sweet potato cultivars in Europe and the USA. The purple potato 'Kufri Neelkanth' has been released for cultivation in northern India. In Europe, the anthocyanin-rich tomato cultivar 'Sun Black' developed via the introgression of Aft and atv genes has been released. The development of anthocyanin-rich food crops without any significant yield penalty has been due to the use of genetic engineering involving specific transcription factors or gene editing. Anthocyanin-rich food ingredients have the potential of being more nutritious than those devoid of anthocyanins. The inclusion of anthocyanins as a target characteristic in breeding programs can ensure the development of cultivars to meet the nutritional needs for human consumption in the developing world.

A real-time recombinase polymerase amplification assay for fast and accurate detection of Ditylenchus destructor.

Ditylenchus destructor is a plant-parasitic nematode that seriously infests sweet potato crop in China. Thus, fast and accurate detection of D. destructor in soil and plant tissue samples is of great significance. In this study, a real-time recombinase polymerase amplification (RPA) assay was developed for the rapid and accurate detection of D. destructor in various samples. The RPA assay could be easily operated and detected as low as 1/500 individual J4 nematode DNA per reaction in 20 min at 39 °C with high specificity. The assay meets the requirements of rapid detection prior to port quarantine as well as on-site real-time detection and can be applied to detect the parasite in soil and plant samples. The modified gDNA extraction method for a single nematode established in this study significantly reduced the time of detection and improved the applicability of the real-time RPA assay for on-site detection in different environments. The real-time RPA assay to detect D. destructor will be useful for epidemiological investigations in the field as well as for quarantine processes in the sweet potato and potato trade.

Polyploid QTL-seq towards rapid development of tightly linked DNA markers for potato and sweetpotato breeding through whole-genome resequencing.

Potato (Solanum tuberosum L.) and sweetpotato (Ipomoea batatas L.), which are nutritionally and commercially important tuberous crops, possess a perplexing heredity because of their autopolyploid genomes. To reduce cross-breeding efforts for selecting superior cultivars from progenies with innumerable combinations of traits, DNA markers tightly linked to agronomical traits are required. To develop DNA markers, we developed a method for quantitative trait loci (QTL) mapping using whole-genome next-generation sequencing (NGS) in autopolyploid crops. To apply the NGS-based bulked segregant method, QTL-seq was modified. (1) Single parent-specific simplex (unique for one homologous chromosome) single-nucleotide polymorphisms (SNPs), which present a simple segregation ratio in the progenies, were exploited by filtering SNPs by SNP index (allele frequency). (2) Clusters of SNPs, which were inherited unevenly between bulked progenies with opposite phenotypes, especially those with an SNP index of 0 for the bulk that did not display the phenotypes of interest, were explored. These modifications allowed for separate tracking of alleles located on each of the multiple homologous chromosomes. By applying this method, clusters of SNPs linked to the potato cyst nematode resistance H1 gene and storage root anthocyanin (AN) content were identified in tetraploid potato and hexaploid sweetpotato, respectively, and completely linked DNA markers were developed at the site of the presented SNPs. Thus, polyploid QTL-seq is a versatile method that is free from specialized manipulation for sequencing and construction of elaborate linkage maps and facilitates rapid development of tightly linked DNA markers in autopolyploid crops, such as potato and sweetpotato.

The Content of Phenolic Acids and Flavonols in the Leaves of Nine Varieties of Sweet Potatoes (Ipomoea batatas L.) Depending on Their Development, Grown in Central Europe.

The aim of the study was the qualitative and quantitative analysis of the bioactive components present in the leaves of 9 sweet potato cultivars grown in the moderate climate in Poland, which were harvested at different growth stages according to the BBCH (Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie) scale (14, 51, 89). It was found that sweet potato leaves contained 7 polyphenolic compounds, including 5 chlorogenic acids-neochlorogenic acid (5-CQA), chlorogenic acid (3-CQA), 4-cryptochlorogenic acid (4-CQA), 34-di-O-caffeoylqunic acid (3,4-CQA), 3,5-di-O-caffeoylqunic acid (3,5-CQA)-and 2 flavonoids, quercetin-3-O-galactoside (Q-3-GA) and quercetin-3-O-glucoside (Q-3-GL). Their content depended on the genotype of the examined cultivars and on the stage of leaf development. The mean content of the identified polyphenolic compounds in the examined cultivars ranged from 148.2 to 14.038.6 mg/100 g-1 DM for the leaves harvested at growth stage 14 according to the BBCH scale. In the case of leaves harvested at BBCH stage 51, the concentration of polyphenolic compounds ranged from 144.76 to 5026.8 mg/100 g-1 DM and at BBCH stage 89 from 4078.1 to 11.183.5 mg/100 g-1 DM. The leaves of the Carmen Rubin cultivar collected at stage 14 contained the highest amount of polyphenolic compounds, while Okinava leaves had the highest amount of these compounds at stage 51. The highest content of polyphenolic compounds in leaves at BBCH growth stage 89 was found in the Radiosa variety. The highest concentration levels were found for 3-CQA at all stages of leaf development. Significant correlations between polyphenol content and antioxidant activity measured by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing/antioxidant power (FRAP) were found. The results of this experiment revealed that the growth stages and genetic properties of cultivars have a very significant influence on the content of phenolic acids and flavonols in sweet potato leaves. The results are innovative and can have a practical application, as the knowledge of the content of the substances under study makes it possible to determine the optimal management practice of sweet potato leaf harvest in order to obtain more top-quality raw material.

Improving the Dietary Vitamin A Content of Rural Communities in South Africa by Replacing Non-Biofortified white Maize and Sweet Potato with Biofortified Maize and Sweet Potato in Traditional Dishes.

Biofortification of staple crops has a potential for addressing micronutrient deficiencies, such as vitamin A deficiency (VAD), which are prevalent in South Africa. The poor acceptability of provitamin A (PVA)-biofortified foods could be improved by combining them with other food items to produce modified traditional dishes. The nutritional composition of the dishes could also be improved by the modification. The study aimed to investigate the effect of replacing white maize and cream-fleshed sweet potato (CFSP)] with PVA-biofortified maize and orange-fleshed sweet potato (OFSP) on the nutritional composition of South African traditional dishes. The protein, fibre, total mineral (ash), lysine, and iron concentrations of the PVA maize phutu (traditional porridge) composite dishes (control), were not significantly different (P > 0.05) from those of white maize phutu composite dishes. However, the PVA concentration of PVA maize phutu composite dishes was higher than that of the white phutu composite dishes (P > 0.05). The OFSP had a significantly lower protein concentration, but a significantly higher (P > 0.05) fibre, ash, lysine, isoleucine, leucine, and PVA concentration, relative to the CFSP. The findings indicate that composite dishes in which white maize is replaced with PVA-biofortified maize, and switching over from CFSP to OFSP, would contribute to combating VAD in South Africa, and in other developing counties.

Change in nuclear DNA content and pollen size with polyploidisation in the sweet potato (Ipomoea batatas, Convolvulaceae) complex.

Genome size evolution and its relationship with pollen grain size has been investigated in sweet potato (Ipomoea batatas), an economically important crop which is closely related to diploid and tetraploid species, assessing the nuclear DNA content of 22 accessions from five Ipomoea species, ten sweet potato varieties and two outgroup taxa. Nuclear DNA amounts were determined using flow cytometry. Pollen grains were studied using scanning and transmission electron microscopy. 2C DNA content of hexaploid I. batatas ranged between 3.12-3.29 pg; the mean monoploid genome size being 0.539 pg (527 Mbp), similar to the related diploid accessions. In tetraploid species I. trifida and I. tabascana, 2C DNA content was, respectively, 2.07 and 2.03 pg. In the diploid species closely related to sweet potato e.g. I. ×leucantha, I. tiliacea, I. trifida and I. triloba, 2C DNA content was 1.01-1.12 pg. However, two diploid outgroup species, I. setosa and I. purpurea, were clearly different from the other diploid species, with 2C of 1.47-1.49 pg; they also have larger chromosomes. The I. batatas genome presents 60.0% AT bases. DNA content and ploidy level were positively correlated within this complex. In I. batatas and the more closely related species I. trifida, the genome size and ploidy levels were correlated with pollen size. Our results allow us to propose alternative or complementary hypotheses to that currently proposed for the formation of hexaploid Ipomoea batatas.

Chemical Constituents of Sweetpotato Genotypes in Relation to Textural Characteristics of Processed French Fries.

Sweetpotato French fries (SPFF) are growing in popularity, however limited information is available on SPFF textural properties in relation to chemical composition. This study investigated the relationship between chemical components of different sweetpotato varieties and textural characteristics of SPFF. Sixteen sweetpotato genotypes were evaluated for (1) chemical constituents; (2) instrumental and sensory textural properties of SPFF; and (3) the relationship between chemical components, instrumental measurements, and sensory attributes. Dry matter (DM), alcohol-insoluble solids (AIS), starch, sugar, and oil content, and also α- and ß-amylase activities were quantified in raw sweetpotatoes and SPFF. Peak force and overall hardness describing instrumental textural properties of SPFF were measured using a texture analyzer. Descriptive sensory analysis was conducted and 10 attributes were evaluated by a trained panel. Results showed that DM, AIS, and starch content in raw sweetpotatoes were significantly correlated (P < 0.05) with instrumental peak force and overall hardness (r = 0.41 to 0.68), and with sensory surface roughness, hardness, fracturability, and crispness (r = 0.63 to 0.90). Total sugar content in raw sweetpotatoes was positively correlated with sensory smoothness and moistness (r = 0.77), and negatively correlated with instrumental peak force and overall hardness (r = -0.62 to -0.69). Instrumental measurements were positively correlated with sensory attributes of hardness, fracturability, and crispness (r = 0.68 to 0.96) and negatively correlated with oiliness, smoothness, moistness, and cohesiveness (r = -0.61 to -0.91). Therefore, DM, AIS, starch, and total sugar contents and instrumental measurements could be used as indicators to evaluate sweetpotato genotypes for SPFF processing. PRACTICAL APPLICATION: In recent years, sweetpotato French fries (SPFF) have grown in popularity, but limited information is available on SPFF textural properties in relation to the differences in chemical constituents among sweetpotato varieties. This study demonstrated that sensory texture attributes of SPFF varied widely and were significantly correlated with chemical components such as dry matter, starch, and total sugar contents of raw sweetpotatoes and instrumental texture measurements of SPFF. The knowledge generated from this study will benefit the food industry and breeding programs with the selection of sweetpotato varieties for improved SPFF quality.

Profiling of anthocyanins in transgenic purple-fleshed sweet potatoes by HPLC-MS/MS.

BACKGROUND: Anthocyanins in purple-fleshed sweet potato (PSP) are beneficial to human health. The leaf color (Lc) gene is a transcription factor involved in regulating anthocyanin biosynthesis. The anthocyanin profiles of wild-type PSP of Ayamurasaki and its three Lc-transgenic lines were investigated by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). In vitro antioxidant activities of wild-type and Lc-transgenic lines, including reducing power activity, DPPH radical scavenging activity, hydroxyl radical scavenging activity, linoleic acid autoxidation inhibition activity, ABTS free radical scavenging activity and oxygen radical absorbance capacity activity, were measured. RESULTS: The results showed that the total anthocyanin contents increased 1.5-1.9 times in three transgenic lines compared with that in wild-type PSP. Seventeen anthocyanins were found in wild-type PSP, while 19 in Lc-transgenic lines including cyanidin-based, peonidin-based and pelargonidin-based anthocyanins. Three pelargonidin-based anthocyanins were detected in three Lc-transgenic lines. Among them, the relative contents of cyanidin-based and pelargonidin-based anthocyanins increased 1.9-2.0 and 3.4-4.5 times respectively, while peonidin-based anthocyanins decreased 1.8-1.9 times in Lc-transgenic lines, compared with wild-type PSP. PSP from wild-type Ayamurasaki and three Lc-transgenic lines exhibited potent antioxidant activities, whereas there was no distinct difference among them. CONCLUSION: The transgene Lc significantly increased the content of total anthocyanins and remarkably changed the anthocyanin profiles in Ayamurasaki. Such novel and high content of anthocyanins obtained in the Lc-transgenic lines with potent antioxidant activities may provide unique functional products with potential helpful for human health. © 2017 Society of Chemical Industry.

Ectopic expression of amaranth seed storage albumin modulates photoassimilate transport and nutrient acquisition in sweetpotato.

Storage proteins in plants, because of high nutrient value, have been a subject of intensive investigation. These proteins are synthesized de novo in the cytoplasm and transported to the storage organelles where they serve as reservoir of energy and supplement of nitrogen during rapid growth and development. Sweetpotato is the seventh most important food crop worldwide, and has a significant contribution to the source of nutrition, albeit with low protein content. To determine the behaviour of seed storage proteins in non-native system, a seed albumin, AmA1, was overexpressed in sweetpotato with an additional aim of improving nutritional quality of tuber proteins. Introduction of AmA1 imparted an increase in protein and amino acid contents as well as the phytophenols. The proteometabolomics analysis revealed a rebalancing of the proteome, with no significant effects on the global metabolome profile of the transgenic tubers. Additionally, the slower degradation of starch and cellulose in transgenic tubers, led to increased post-harvest durability. Present study provides a new insight into the role of a seed storage protein in the modulation of photoassimilate movement and nutrient acquisition.

Expressing the sweet potato orange gene in transgenic potato improves drought tolerance and marketable tuber production.

Potato (Solanum tuberosum L.) is generally considered to be sensitive to drought stress. Even short periods of water shortage can result in reduced tuber production and quality. We previously reported that transgenic potato plants expressing the sweet potato orange gene (IbOr) under the control of the stress-inducible SWPA2 promoter (referred to as SOR plants) showed increased tolerance to methyl viologen-mediated oxidative stress and high salinity, along with increased carotenoid contents. In this study, in an effort to improve the productivity and environmental stress tolerance of potato, we subjected transgenic potato plants expressing IbOr to water-deficient conditions in the greenhouse. The SOR plants exhibited increased tolerance to drought stress under greenhouse conditions. IbOr expression was associated with slightly negative phenotypes, including reduced tuber production. Controlling IbOr expression imparted the same degree of drought tolerance while ameliorating these negative phenotypic effects, leading to levels of tuber production similar to or better than those of wild-type plants under drought stress conditions. In particular, under drought stress, drought tolerance and the production of marketable tubers (over 80g) were improved in transgenic plants compared with non-transgenic plants. These results suggest that expressing the IbOr transgene can lead to significant gains in drought tolerance and tuber production in potato, thereby improving these agronomically important traits.

Phytochemical changes in phenolics, anthocyanins, ascorbic acid, and carotenoids associated with sweetpotato storage and impacts on bioactive properties.

Sweetpotato phytochemical content was evaluated in four genotypes (NCPUR06-020, Covington, Yellow Covington, and NC07-847) at harvest and after curing/storage for 4 or 8 months. Curing and storage for up to 8 months did not significantly affect total phenolic content in Covington, Yellow Covington, and NC07-847, however for NCPUR06-020, a purple-fleshed selection, total phenolic content declined mainly due to anthocyanin degradation during storage. Covington had the highest carotenoid content at harvest time (281.9 µg/g DM), followed by NC07-847 (26.2 µg/g DM), and after 8 months, total carotenoids had increased by 25% and 50%, respectively. Antioxidant activity gradually declined during storage, and freshly harvested sweetpotatoes also demonstrated higher anti-inflammatory capacity as gauged by inhibition of lipopolysaccharide-induced reactive oxygen species (ROS) in SH-SY5Y cells. Gradual changes in sweetpotato phytochemical content and antioxidant and anti-inflammatory capacity were noted during normal long-term storage, but the specific effects were genotype-dependent.

Cloning and characterization of an Orange gene that increases carotenoid accumulation and salt stress tolerance in transgenic sweetpotato cultures.

The Orange (Or) gene is responsible for the accumulation of carotenoids in plants. We isolated the Or gene (IbOr) from storage roots of orange-fleshed sweetpotato (Ipomoea batatas L. Lam. cv. Sinhwangmi), and analyzed its function in transgenic sweetpotato calli. The IbOr gene has an open reading frame in the 942 bp cDNA, which encodes a 313-amino acid protein containing a cysteine-rich zinc finger domain. IbOr was strongly expressed in storage roots of orange-fleshed sweetpotato cultivars; it also was expressed in leaves, stems, and roots of cultivars with alternatively colored storage roots. IbOr transcription increased in response to abiotic stress, with gene expression reaching maximum at 2 h after treatment. Two different overexpression vectors of IbOr (IbOr-Wt and IbOr-Ins, which contained seven extra amino acids) were transformed into calli of white-fleshed sweetpotato [cv. Yulmi (Ym)] using Agrobacterium. The transgenic calli were easily selected because they developed a fine orange color. The expression levels of the IbOr transgene and genes involved in carotenoid biosynthesis in IbOr-Wt and IbOr-Ins transgenic calli were similar, and both transformants displayed higher expression levels than those in Ym calli. The contents of ß-carotene, lutein, and total carotenoids in IbOr-Ins transgenic lines were approximately 10, 6, and 14 times higher than those in Ym calli, respectively. The transgenic IbOr calli exhibited increased antioxidant activity and increased tolerance to salt stress. Our work shows that the IbOr gene may be useful for the biotechnological development of transgenic sweetpotato plants that accumulate increased carotenoid contents on marginal agricultural lands.

Historical collections reveal patterns of diffusion of sweet potato in Oceania obscured by modern plant movements and recombination.

The history of sweet potato in the Pacific has long been an enigma. Archaeological, linguistic, and ethnobotanical data suggest that prehistoric human-mediated dispersal events contributed to the distribution in Oceania of this American domesticate. According to the "tripartite hypothesis," sweet potato was introduced into Oceania from South America in pre-Columbian times and was then later newly introduced, and diffused widely across the Pacific, by Europeans via two historically documented routes from Mexico and the Caribbean. Although sweet potato is the most convincing example of putative pre-Columbian connections between human occupants of Polynesia and South America, the search for genetic evidence of pre-Columbian dispersal of sweet potato into Oceania has been inconclusive. Our study attempts to fill this gap. Using complementary sets of markers (chloroplast and nuclear microsatellites) and both modern and herbarium samples, we test the tripartite hypothesis. Our results provide strong support for prehistoric transfer(s) of sweet potato from South America (Peru-Ecuador region) into Polynesia. Our results also document a temporal shift in the pattern of distribution of genetic variation in sweet potato in Oceania. Later reintroductions, accompanied by recombination between distinct sweet potato gene pools, have reshuffled the crop's initial genetic base, obscuring primary patterns of diffusion and, at the same time, giving rise to an impressive number of local variants. Moreover, our study shows that phenotypes, names, and neutral genes do not necessarily share completely parallel evolutionary histories. Multidisciplinary approaches, thus, appear necessary for accurate reconstruction of the intertwined histories of plants and humans.

Enhanced salt stress tolerance in transgenic potato plants expressing IbMYB1, a sweet potato transcription factor.

IbMYB1, a transcription factor (TF) for R2R3-type MYB TFs, is a key regulator of anthocyanin biosynthesis during storage of sweet potatoes. Anthocyanins provide important antioxidants of nutritional value to humans, and also protect plants from oxidative stress. This study aimed to increase transgenic potatoes' (Solanum tuberosum cv. LongShu No.3) tolerance to environmental stress and enhance their nutritional value. Transgenic potato plants expressing IbMYB1 genes under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter (referred to as SM plants) were successfully generated through Agrobacterium-mediated transformation. Two representative transgenic SM5 and SM12 lines were evaluated for enhanced tolerance to salinity, UV-B rays, and drought conditions. Following treatment of 100 mM NaCl, seedlings of SM5 and SM12 lines showed less root damage and more shoot growth than control lines expressing only an empty vector. Transgenic potato plants in pots treated with 400 mM NaCl showed high amounts of secondary metabolites, including phenols, anthocyanins, and flavonoids, compared with control plants. After treatment of 400 mM NaCl, transgenic potato plants also showed high DDPH radical scavenging activity and high PS II photochemical efficiency compared with the control line. Furthermore, following treatment of NaCl, UV-B, and drought stress, the expression levels of IbMYB1 and several structural genes in the flavonoid biosynthesis such as CHS, DFR, and ANS in transgenic plants were found to be correlated with plant phenotype. The results suggest that enhanced IbMYB1 expression affects secondary metabolism, which leads to improved tolerance ability in transgenic potatoes.

Molecular cloning and functional analysis of the ortho-hydroxylases of p-coumaroyl coenzyme A/feruloyl coenzyme A involved in formation of umbelliferone and scopoletin in sweet potato, Ipomoea batatas (L.) Lam.

Ortho-hydroxylation of cinnamates is a key step in coumarin biosynthesis in plants. Ortho-hydroxylated cinnamates undergo trans/cis isomerization of the side-chain and then lactonization to form coumarins. Sweet potato [Ipomoea batatas (L.) Lam.] accumulates umbelliferone and scopoletin after biotic and abiotic stresses. To elucidate molecular aspects of ortho-hydroxylation involved in umbelliferone formation in sweet potato, isolation and characterization of cDNAs encoding 2-oxoglutarate-dependent dioxygenases (2OGD) was performed from sweet potato tubers treated with a chitosan elicitor. Five cDNAs (designated as Ib) encoding a protein of 358 amino acid residues were cloned, and these were categorized into two groups, Ib1 and Ib2, based on their amino acid sequences. Whether the recombinant Ib proteins had any enzymatic activity toward cinnamates was examined. Ib1 proteins exhibited ortho-hydroxylation activity toward feruloyl coenzyme A (CoA) to form scopoletin (K(m)=~10 µM, k(cat)=~2.7s(-1)). By contrast, Ib2 proteins catalyzed ortho-hydroxylation of feruloyl-CoA (K(m)=7.3-14.0 µM, k(cat)=0.28-0.55 s(-1)) and also of p-coumaroyl-CoA (K(m)=6.1-15.2 µM, k(cat)=0.28-0.64 s(-1)) to form scopoletin and umbelliferone, respectively. Fungal and chitosan treatments increased levels of umbelliferone and its glucoside (skimmin) in the tubers, and expression of the Ib2 gene was induced concomitantly.

Expression of a cloned sweet potato catalase SPCAT1 alleviates ethephon-mediated leaf senescence and H2O2 elevation.

In this report a full-length cDNA, SPCAT1, was isolated from ethephon-treated mature L3 leaves of sweet potato. SPCAT1 contained 1479 nucleotides (492 amino acids) in its open reading frame, and exhibited high amino acid sequence identities (ca. 71.2-80.9%) with several plant catalases, including Arabidopsis, eggplant, grey mangrove, pea, potato, tobacco and tomato. Gene structural analysis showed that SPCAT1 encoded a catalase and contained a putative conserved internal peroxisomal targeting signal PTS1 motif and calmodulin binding domain around its C-terminus. RT-PCR showed that SPCAT1 gene expression was enhanced significantly in mature L3 and early senescent L4 leaves and was much reduced in immature L1, L2 and completely yellowing senescent L5 leaves. In dark- and ethephon-treated L3 leaves, SPCAT1 expression was significantly enhanced temporarily from 0 to 24h, then decreased gradually until 72h after treatment. SPCAT1 gene expression levels also exhibited approximately inverse correlation with the qualitative and quantitative H(2)O(2) amounts. Effector treatment showed that ethephon-enhanced SPCAT1 expression was repressed by antioxidant reduced glutathione, NADPH oxidase inhibitor diphenylene iodonium (DPI), calcium ion chelator EGTA and de novo protein synthesis inhibitor cycloheximide. These data suggest that elevated reactive oxygen species H(2)O(2), NADPH oxidase, external calcium influx and de novo synthesized proteins are required and associated with ethephon-mediated enhancement of sweet potato catalase SPCAT1 expression. Exogenous application of expressed catalase SPCAT1 fusion protein delayed or alleviated ethephon-mediated leaf senescence and H(2)O(2) elevation. Based on these data we conclude that sweet potato SPCAT1 is an ethephon-inducible peroxisomal catalase, and its expression is regulated by reduced glutathione, DPI, EGTA and cycloheximide. Sweet potato catalase SPCAT1 may play a physiological role or function in cope with H(2)O(2) homeostasis in leaves caused by developmental cues and environmental stimuli.

A sweetpotato SRD1 promoter confers strong root-, taproot-, and tuber-specific expression in Arabidopsis, carrot, and potato.

Harvestable, starch-storing organs of plants, such as fleshy taproots and tubers, are important agronomic products that are also suitable target organs for use in the molecular farming of recombinant proteins due to their strong sink strength. To exploit a promoter directing strong expression restricted to these storage organs, we isolated the promoter region (3.0 kb) of SRD1 from sweetpotato (Ipomoea batatas cv. 'White Star') and characterized its activity in transgenic Arabidopsis, carrot, and potato using the ß-glucuronidase (GUS) gene (uidA) as a reporter gene. The SRD1 promoter conferred root-specific expression in transgenic Arabidopsis, with SRD1 promoter activity increasing in response to exogenous IAA. A time-course study of the effect of IAA (50 µM) revealed a maximum increase in SRD1 promoter activity at 24 h post-treatment initiation. A serial 5' deletion analysis of the SRD1 promoter identified regions related to IAA-inducible expression as well as regions containing positive and negative elements, respectively, controlling the expression level. In transgenic carrot, the SRD1 promoter mediated strong taproot-specific expression, as evidenced by GUS staining being strong in almost the entire taproot, including secondary phloem, secondary xylem and vascular cambium. The activity of the SRD1 promoter gradually increased with increasing diameter of the taproot in the transgenic carrot and was 10.71-fold higher than that of the CaMV35S promoter. The SRD1 promoter also directed strong tuber-specific expression in transgenic potato. Taken together, these results demonstrate that the SRD1 promoter directs strong expression restricted to the underground storage organs, such as fleshy taproots and tubers, as well as fibrous root tissues.