Production of Agrocinopine A by Ipomoea batatas Agrocinopine Synthase in Transgenic Tobacco and Its Effect on the Rhizosphere Microbial Community.

Agrobacterium tumefaciens is a bacterial pathogen that causes crown gall disease on a wide range of eudicot plants by genetic transformation. Besides T-DNA integrated by natural transformation of plant vegetative tissues by pathogenic Agrobacterium spp., previous reports have indicated that T-DNA sequences originating from an ancestral Agrobacterium sp. are present in the genomes of all cultivated sweet potato (Ipomoea batatas) varieties analyzed. Expression of an Agrobacterium-derived agrocinopine synthase (ACS) gene was detected in leaf and root tissues of sweet potato, suggesting that the plant can produce agrocinopine, a sugar-phosphodiester opine considered to be utilized by some strains of Agrobacterium spp. in crown gall. To validate the product synthesized by Ipomoea batatas ACS (IbACS), we introduced IbACS into tobacco under a constitutive promoter. High-voltage paper electrophoresis followed by alkaline silver nitrate staining detected the production of an agrocinopine-like substance in IbACS1-expressing tobacco, and further mass spectrometry and nuclear magnetic resonance analyses of the product confirmed that IbACS can produce agrocinopine A from natural plant substrates. The partially purified compound was biologically active in an agrocinopine A bioassay. A 16S ribosomal RNA amplicon sequencing and meta-transcriptome analysis revealed that the rhizosphere microbial community of tobacco was affected by the expression of IbACS. A new species of Leifsonia (actinobacteria) was isolated as an enriched bacterium in the rhizosphere of IbACS1-expressing tobacco. This Leifsonia sp. can catabolize agrocinopine A produced in tobacco, indicating that the production of agrocinopine A attracts rhizosphere bacteria that can utilize this sugar-phosphodiester. These results suggest a potential role of IbACS conserved among sweet potato cultivars in manipulating their microbial community.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Carbohydrate components in sweetpotato storage roots: their diversities and genetic improvement.

Carbohydrates are important components in sweetpotatoes in terms of both their industrial use and eating quality. Although there has been a narrow range of diversity in the properties of sweetpotato starch, unique varieties and experimental lines with different starch traits have been produced recently both by conventional breeding and genetic engineering. The diversity in maltose content, free sugar composition and textural properties in sweetpotato cultivars is also important for their eating quality and processing of storage roots. In this review, we summarize the current status of research on and breeding for these important traits and discuss the future prospects for research in this area.

Molecular characterization of genes encoding isoamylase-type debranching enzyme in tuberous root of sweet potato, Ipomoea batatas (L.) Lam.

Isoamylase (ISA) is a starch debranching enzyme that removes α-1,6-glucosidic linkages in α-polyglucans such as amylopectin. From previous studies, plant isoamylases have been shown to play a crucial role in amylopectin biosynthesis; however, little is known about their function in storage root tissues of plants such as cassava, yam and sweet potato. In this study, we isolated cDNA clones and characterized the cDNA nucleotide sequences of three genes (IbISA1, IbISA2, IbISA3) encoding isoamylase from sweet potato (Ipomoea batatas (L.) cv. White Star). Deduced amino acid sequences of the three isolated IbISAs have the specific regions that are highly conserved among the α-amylase family members. The product of IbISA2 is predicted to be enzymatically inactive, like other plant ISA2s, due to replacement of amino acid residues that are important for hydrolytic reaction. qRT-PCR analysis demonstrated that expression of IbISA2 was higher than that of the other two IbISAs (IbISA1 and IbISA3) in tuberous root at 109 days after planting, at which stage of tuberous root was at which stage tuberous roots were almost fully developed almost developed. This expression pattern observed in our experiments was different from that in other sink organs, such as seeds (endosperms), indicating that orchestration of ISA gene expression may depend on the differences in sink organ type between tuberous roots and seeds. The molecular characterization of three IbISA genes and their expression analysis in this study will contribute to further studies on starch biosynthesis in sweet potato, especially in storage root.

Inhibition of the expression of the starch synthase II gene leads to lower pasting temperature in sweetpotato starch.

The sweetpotato cultivar Quick Sweet (QS) with a lower pasting temperature of starch is a unique breeding material, but the biochemical background of this property has been unknown. To assess the physiological impact of the reduced isoform II activity of starch synthase (SSII) on the starch properties in sweetpotato storage root, transgenic sweetpotato plants with reduced expressions of the SSII gene were generated and evaluated. All of the starches from transgenic plants showed lower pasting temperatures and breakdown measured by a Rapid Visco Analyzer. The pasting temperatures in transgenic plants were approximately 10-15 degrees C lower than in wild-type plants. Distribution of the amylopectin chain length of the transgenic lines showed marked differences compared to that in wild-type plants: more chains with degree of polymerization (DP) 6-11 and fewer chains with DP 13-25. The starch granules from the storage root of transgenic plants showed cracking on the hilum, while those from wild-type plants appeared to be typical sweetpotato starch. In accordance with these observations, the expression of SSII in the storage roots of the sweetpotato cultivar with low pasting temperature starch (QS) was notably lower than in cultivars with normal starch. Moreover, nucleotide sequence analysis suggested that most of the SSII transcripts in the cultivar with low pasting temperature starch were inactive alleles. These results clearly indicate that the activity of SSII in sweetpotato storage roots, like those in other plants, affects the pasting properties of starch through alteration of the amylopectin structure.

Cloning and characterization of a squalene synthase gene from a petroleum plant, Euphorbia tirucalli L.

Euphorbia tirucalli L., which is also known as a petroleum plant, produces a large amount of phytosterols and triterpenes. During their biosynthesis, squalene synthase converts two molecules of the hydrophilic substrate farnesyl diphosphate into a hydrophobic product, squalene. An E. tirucalli cDNA clone of a putative squalene synthase gene (EtSS) was isolated by RT-PCR followed by 5'- and 3'-RACE. The restriction fragment polymorphisms revealed by Southern blot analysis suggest that EtSS is a single copy gene. The glycine at the 287th residue from the N-terminal end of domain C has replaced alanine, which is conserved among all the other SS sequences deposited in the Genbank database. The N-terminal 380 residues of the hydrophilic sequence was expressed as a peptide-tagged protein in E. coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. E. tirucalli transgenic callus lines, in which EtSS was overexpressed, accumulated increased amounts of phytosterols as compared with that of wild type callus. RT-PCR analysis of wild type E. tirucalli plants revealed that the EtSS transcript accumulated in almost equal amounts in the stems and the leaves with a stalk, while a lower amount was detected in the roots. In situ hybridization analysis revealed that prominent antisense-probe signal was detected in the cambia within bundle sheathes. These results indicate that EtSS functions prominently in cambia, which are located adjacent to conductive tubes, and that this gene plays important roles in phytosterol accumulation in petroleum plants.

Expression of the gene for sterol-biosynthesis enzyme squalene epoxidase in parenchyma cells of the oil plant, Euphorbia tirucalli.

In plants, phytosterols and triterpenes are major secondary metabolites. In an attempt to reveal the mechanism for synthesis and storage of these compounds, we isolated and characterized cDNA clones for squalene epoxidase (SE), from a succulent shrub, Euphorbia tirucalli. Southern-blot analysis of total DNA using cDNA fragment as a probe showed that the E. tirucalli squalene epoxidase gene (EtSE) is single-copy type in terms of restriction fragment length polymorphism (RFLP). Deduced amino-acid sequence of the cDNA showed 83 and 75% identity to those of rice and ginseng, respectively, in an area excluding a less homologous putative transmembrane region in the N-terminal end. Functional characterization with heterologous expression using an erg1-disrupted yeast mutant KLN1 indicated that the EtSE recovered ergosterol auxotrophy of the mutant, and gave rise to an ergosterol accumulation in the EtSE transformant. RT-PCR analysis showed the EtSE transcripts in leaves and stem internodes accumulated in almost equal amounts, which were more abundant than those in roots. In situ hybridization using EtSE antisense probe revealed prominent EtSE expression on a parenchyma cell adjacent to primary laticifers that were located in a rosary orientation in the inner region of cortex. This is the first report of expression of a gene for a rate-limiting enzyme in mevalonate pathway in organs and tissues of a plant.