Absence of amylose in sweet potato [Ipomoea batatas (L.) Lam.] following the introduction of granule-bound starch synthase I cDNA

The full-length sense cDNA for sweet potato granule-bound starch synthase I (GBSSI) driven by the CaMV 35S promoter was introduced into the sweet potato by Agrobacterium tumefaciens-mediated transformation. Out of the 26 transgenic plants obtained, one plant showed the absence of amylose in the tuberous root as determined by the iodine colorimetric method. Electrophoresis analysis failed to detect the GBSSI protein, suggesting that gene silencing of the GBSSI gene occurred in the transgenic sweet potato plant. These results demonstrate that starch composition in the tuberous root of sweet potato can be altered by genetic transformation.     

Effects of single and mixed inoculation with two arbuscular mycorrhizal fungi in two different levels of phosphorus supply on β-carotene concentrations in sweet potato (Ipomoea batatas L.) tubers

Aims

This study aimed to determine the effect of arbuscular mycorrhizal (AM) fungi and phosphorus (P) supply levels on β-carotene concentrations in sweet potato (Ipomoea batatas L.) tubers.

Methods

Two commercial AM fungal isolates of Glomus intraradices (IFP Glintra) and Glomus mosseae (IFP Glm) which differ in their life cycles were used. Sweet potato plants were grown in a horizontal split-root system that consisted of two root compartments. A root-free fungal compartment that allowed the quantification of mycelial development was inserted into each root compartment. The two root compartments were inoculated either with the same or with different AM isolates, or remained free of mycorrhizal propagules. Each fungal treatment was carried out in two P supply levels.

Results

In the low P supply level, mycorrhizal colonization significantly increased β-carotene concentrations in sweet potato tubers compared with the non-mycorrhizal plants. Glomus intraradices appeared to be more efficient in increasing β-carotene concentrations than G. mosseae. Dual inoculation of the root system with the two mycorrhizal fungi did not result in a higher increase in tuber β-carotene concentrations than inoculation with the single isolates. Improved P nutrition led to higher plant tuber biomass but was not associated with increased β-carotene concentrations.

Conclusions

The results indicate a remarkable potential of mycorrhizal fungi to improve β-carotene concentrations in sweet potato tubers in low P fertilized soils. These results also suggest that β-carotene metabolism in sweet potato tubers might be specifically activated by root mycorrhizal colonization.     

Starch self-processing in transgenic sweet potato roots expressing a hyperthermophilic α-amylase

Sweet potato is a major crop in the southeastern United States, which requires few inputs and grows well on marginal land. It accumulates large quantities of starch in the storage roots and has been shown to give comparable or superior ethanol yields to corn per cultivated acre in the southeast. Starch conversion to fermentable sugars (i.e., for ethanol production) is carried out at high temperatures and requires the action of thermostable and thermoactive amylolytic enzymes. These enzymes are added to the starch mixture impacting overall process economics. To address this shortcoming, the gene encoding a hyperthermophilic α-amylase from Thermotoga maritima was cloned and expressed in transgenic sweet potato, generated by Agrobacterium tumefaciens-mediated transformation, to create a plant with the ability to self-process starch. No significant enzyme activity could be detected below 40°C, but starch in the transgenic sweet potato storage roots was readily hydrolyzed at 80°C. The transgene did not affect normal storage root formation. The results presented here demonstrate that engineering plants with hyperthermophilic glycoside hydrolases can facilitate cost effective starch conversion to fermentable sugars. Furthermore, the use of sweet potato as an alternative near-term energy crop should be considered.     

Unexpected mode of action of sweet potato β-amylase on maltooligomer substrates

β-Amylase (EC 3.2.1.2), one of the main protein of the sweet potato, is an exo-working enzyme catalyzing the hydrolysis of α(1,4) glycosidic linkages in polysaccharides and removes successively maltose units from the non-reducing ends. The enzyme belongs to glycoside hydrolase GH14 family and inverts the anomeric configuration of the hydrolysis product. Multiple attack or processivity is an important property of polymer active enzymes and there is still limited information about the processivity of carbohydrate active enzymes. Action pattern and kinetic measurements of sweet potato β-amylase were made on a series of aromatic chromophor group-containing substrates (degree of polymerization DP 3-13) using HPLC method. Measured catalytic efficiencies increased with increasing DP of the substrates. Processive cleavage was observed on all substrates except the shortest pentamer. The mean number of steps without dissociation of enzyme–product complex increases with DP of substrate and reached 3.3 in case of CNPG11 indicating that processivity on longer substrates was more significant. A unique transglycosylation was observed on those substrates, which suffer processive cleavage and the substrates were re-built by the enzyme. Our results are the first presentation of a transglycosylation during an inverting glycosidase catalyzed hydrolysis. The yield of transglycosylation was remarkable high as shown in the change of the CNPG11 quantity. The CNPG11 concentration was doubled (from 0.24 to 0.54 mM) in the early phase of the reaction.     

不同碳源对甘薯(Ipomoea batatas L.Lam.)块根愈伤组织的形成和生长的影响

甘薯块根愈伤组织的形成中,蔗糖,可溶性淀粉和葡萄糖作为碳源效果最好,麦芽糖和果糖次之,木糖仅形成少量的愈伤组织,而半乳糖不能被利用。在愈伤组织的生长上,葡萄糖和蔗糖效果最好,果糖和淀粉次之,木糖和半乳糖均不能被利用。愈伤组织最适生长的蔗糖和葡萄糖浓度为2％左右,浓度增加到8％时,生长几乎全部被抑制。无碳源时,仅形成少量的愈伤组织,但不能促进生长。 愈伤组织中过氧化物酶的同工酶的谱带与块根组织的谱带相同,但前者酶活性较强。不同碳源培养的愈伤组织的酶谱也相同。 以蔗糖和葡萄糖培养时,愈伤组织中可溶性糖含量较高,而其他碳源培养的组织则含量较低。淀粉在麦芽糖和果糖培养的愈伤组织中含量较高,而以其他碳源培养的组织含量较低。     

Molecular cloning and characterization of a flavonoid 3′-hydroxylase gene from purple-fleshed sweet potato (Ipomoea batatas)

Flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21) is an important enzyme which determines the hydroxylation pattern of anthocyanins. In this study, the full-length cDNA and genomic DNA of F3′H were isolated and characterized from the purple-fleshed sweet potato (Ipomoea batatas). IbF3’′H was 1,789 bp containing a 1,554 bp open reading frame (ORF) encoding 518 amino acids. Comparative and bioinformatic analysis revealed that IbF3′H was highly homologous with F3′Hs from other plant species. Conserved domain search revealed that IbF3′H was a cytochrome P450 dependent enzyme. Three F3′H-specific motifs (V₇₅VVAAS₈₀, G₄₂₇GEK₄₃₀ and V₄₃₃DVKG₄₃₇) were conserved in IbF3′H. Phylogenetic analysis revealed that IbF3′H was clustered into the same subgroup with the homologues from I. purpurea, I. tricolor and I. nil. There were multiple copies of the IbF3′H gene in the genome of I. batatas. IbF3′H was constitutively expressed in all tested tissues including fibrous roots, thick roots, storage roots, stems and leaves. During storage root formation, IbF3′H was expressed most abundantly in the storage roots, suggesting that the anthocyanin biosynthesis is also active in the under-ground organs. IbF3′H expression was associated with anthocyanin accumulation in five different sweet potato cultivars tested. Complementative analysis implied that the full-length cDNA of IbF3′H could encode a functional protein and had a special catalytic activity of flavonoid 3′-hydroxylase.     

甘薯(Ipomoea batatas L.)遗传转化几个因素的研究

目的:提高甘薯的遗传转化率,为建立快速高效的甘薯遗传转化体系奠定基础。方法:以甘薯多个优良栽培品种无菌苗茎切段为受体,利用农杆菌LBA4404/pSP10Z做介导,研究了影响甘薯转化的几个因素。结果:接种侵染时间对转化效率影响很大,以2~15min为宜,最高转化率可达32.14%;不同基因型的转化受体之间转化率差别较大;在培养过程中最后30min加入终浓度为50μmol/L AS的接种菌侵染的外植体,转化率较对照提高了2.02倍;共培养培养基中加入终浓度为50μmol/L的AS,转化率较对照组提高了6.11倍;AS终浓度为50μmol/L同时pH为4.8的共培养培养基有利于转化的发生,转化率较对照组提高了1.73倍;共培养培养基中脯氨酸的添加并不能提高转化率。结论:该研究为快速高效甘薯遗传转化体系的建立提供了依据。     

Phytochrome-mediated regulation of β-amylase mRNA level in mustard (Sinapis alba L.) cotyledons

Phytochrome, activated by continuous red light, increases the amount of total polyadenylated RNA during photomorphogenesis of mustard (Sinapis alba L.) cotyledons. In-vitro translation of total polyadenylated RNA in a reticulocyte translation system has shown that the activity of translatable -amylase mRNA is increased by phytochrome about threefold in the 3-d-old cotyledons, based on equal amounts of polyadenylated RNA, and about eightfold on a per-cotyledon basis. Cordycepin prevents the accumulation of translatable -amylase mRNA. It is concluded that the phytochrome-mediated control of -amylase synthesis is exerted on the level of mRNA synthesis. During seedling development in continuous red light, a phytochrome-dependent increase of -amylase mRNA can be observed at least 6 h before the onset of -amylase synthesis. If, after a period of enzyme synthesis, phytochrome action is interrupted by long-wavelength far-red light followed by darkness, -amylase mRNA as well as -amylase synthesis remain at a high level for 8–10 h and then decline sharply. It is concluded that -amylase mRNA, having an apparent lifetime of the order of 8–10 h, can be formed under the influence of phytochrome during early seedling development but it activates -amylase synthesis only after a lag-phase of about 8 h, when the cotyledons acquire competence to synthesize the enzyme. The consequences of these findings for the signal-transduction chain of phytochrome are discussed.Abbreviations EDTA Na₂-ethylenediaminotetraacetic acid - PAGE polyacrylamide gel electrophoresis - poly(A)⁺RNA polyadenylated mRNA - P_r, P_fr red- and far-red-absorbing forms of phytochrome - SDS sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

A tobacco microsomal ω-3 fatty acid desaturase gene increases the linolenic acid content in transgenic sweet potato (Ipomoea batatas)

A tobacco microsomal P-3 fatty acid desaturase gene (NtFAD3) under the control of the CaMV 35S promoter or an improved CaMV 35S promoter (El2Q) was introduced into sweet potato. Transformed sweet potato plants were obtained from embryogenic calli following Agrobacterium tumefaciens-mediated transformation. The transgenic plants grew normally to form storage roots and showed properties similar to those of the non-transgenic plants. The fatty acid composition in the transgenic line with a NtFAD3 gene driven by the CaMV 35S promoter was similar to that in the non-transformant. However, in the transgenic line that had a NtFAD3 gene driven by the El2Q promoter, linoleic acid (18:2) and linolenic acid (18:3) contents were 47.7 mol% and 24.8 mol%, respectively, which were significantly different from the 53.6 mol% and 11.3 mol%, respectively, in the non-transformant. The NtFAD3 gene driven by the El2Q promoter was expressed more strongly than that driven by the CaMV 35S promoter, thereby increasing the linolenic acid content in the transgenic sweet potato plants.     

The inheritance of β-amylases in developing barley grain

Three types of -amylase isozyme pattern (Sd^d, Sd^e, and Sd^f) were observed in the doughy endosperm stage of ripening barley grains. From crosses between Sd^d and Sd^f types, it was concluded that these phenotypes are controlled by a pair of codominant alleles. Preliminary studies on the distribution of these -amylase zymotypes indicated that this polymorphism in developing grain is related to a -amylase polymorphism in barley malt.     

High-level expression of sucrose inducible sweet potato sporamin gene promoter: β-glucuronidase fusion gene in transgenic Nicotiana plumbaginifolia hairy roots

In Vitro Cellular & Developmental Biology - Plant - We developed transgenic Nicotiana plumbaginifolia hairy roots with sucrose-inducible minimal promoter (Spomin)-β-glucuronidase (GUS)...     

Chemical constituents from the roots of Fallopia convolvulus (L.) A. Löve

Twenty compounds, including three sterols (1–3), three phenols (4, 14 and 15), four anthraquinones (5, 7, 8 and 16), one chromone (6), two stilbenes (9 and 10), three amides (11–13), three flavonoids (17–19) and one organic acid (20), were obtained by modern phytochemical isolation methods. Their structures were identified by spectroscopic methods and in comparison with the published data in the references. Among them, compound 2, 3, 11 and 13 were firstly discovered from genus Fallopia, and compounds 1, 5–8, 10, 14, 15, 17, 19 and 20 were obtained from F. convolvulus for the first time. The chemotaxonomic significance of these compounds was also discussed, which revealed the relationships between F. convolvulus and some other species of Polygonaceae family.     

Sequential control of phytochrome-mediated synthesis de novo of β-amylase in the cotyledons of mustard (Sinapis alba L.) seedlings

In the cotyledons of mustard (Sinapis alba L.) seedlings irradiated from the time of sowing with continuous red light, the photoreversibility of the phytochrome-mediated increase in -amylase activity (EC 3.2.1.2) is lost 36 h after sowing (coupling point). However, the induced increase of -amylase activity cannot be detected before 46 h after sowing (starting point). Density labeling with deuterium oxide shows that the increase of enzyme activity in light and darkness coincides precisely with the synthesis of -amylase protein. Thus, phytochrome mediates an increase of -amylase synthesis de novo. Since there is no turnover detectable by density labeling, it is concluded that -amylase of mustard cotyledons is a physiologically stable enzyme (half-life >5 d). The 10-h time gap between loss of photoreversibility and onset of light-induced -amylase synthesis points to a relatively stable regulatory element within the signal chain (transmitter) which links -amylase synthesis to the primary action of phytochrome. A 12-h lag between the cessation of phytochrome action and the cessation of induced -amylase synthesis indicates a limited lifetime of the transmitter (about 12 h). The effect of this result on the interpretation of the coupling point is discussed.Abbreviations P_r, P_fr red and far-red absorbing forms of phytochrome     

The effects of IAA and tetcyclacis on tuberization in potato (Solanum tuberosum L.) shoot cultures in vitro

Potato (Solanum tuberosum cv. Désirée) shoots grown in vitro in continuous darkness or in long days (LDs), were used to investigate indole-3-acetic acid (IAA) effects on stolon initiation and tuber formation, combining IAA with increased or decreased gibberellin levels. An increased gibberellin (GA) level was achieved by the applying 1 μM GA₃, while decreased gibberellin level was presumably realized by the adding 3 μM tetcyclacis (Tc). About 15% of potato shoots developed stolons both in LDs and in darkness. Stolon initiation was stimulated by GA₃ in darkness and by Tc in LDs. Tuber formation was strongly inhibited in LDs and by GA₃ both in light and darkness, but stimulated in darkness at low GA level. Exceptionally, tuber formation occurred in LDs at the highest Tc concentrations, in about 25% of explants. Indole-3-acetic acid alone stimulated stolon formation in LDs, both in the presence or absence of GA₃. IAA alone also stimulated tuber formation in dark-grown shoots, but could not overcome the inhibitory effect of LDs. Indications that, depending on their concentration ratio, IAA may interact with GA₃ in different tuberization phases, have been discussed. Radomir Konjević—Deceased in July 2006.     

Haplotype diversity in the endosperm specific β-amylase gene Bmy1 of cultivated barley (Hordeum vulgare L.)

Five single nucleotide polymorphism (SNP) sites corresponding to substitutions in the protein sequence of the β-amylase gene Bmy1 at amino acid (AA) positions 115, 165, 233, 347 and 430 were genotyped in 493 cultivated barley accessions by Pyrosequencing and a CAPS assay. A total of 6 different haplotypes for the Bmy1 gene were discovered of which 4 haplotypes were identified as previously described alleles Bmy1-Sd1, Bmy1-Sd2L, Bmy1-Sd2H and Bmy1-Sd3, while 2 haplotypes were new. A broad spectrum of haplotypes was found in spring barleys, while the winter barleys were dominated by the newly described haplotype Bmy1-Sd4. Individual haplotype frequencies varied between the geographic regions.Three pairs of SNP loci within the gene showed highly significant (P<0.0001) elevated values of linkage disequilibrium (LD) with r ² > 0.6. In the European and Asian subpopulations different loci were in linkage disequilibrium due to the differences in haplotype frequency distributions. By applying LD data to select haplotype tagging SNPs, three SNP sites corresponding to AA positions 115, 233 and 347 were identified that allowed to discriminate 4 haplotypes and to capture 91.6% of the available diversity by distinguishing 452 out of 493 accessions. In a subset of 2-rowed German spring barley varieties 4 SNPs and 2 haplotypes had a significant association with the malting quality parameter final attenuation limit which is related to the total amylolytic enzymatic activity.     

Characterisation of the cDNA clones of two β-tubulin genes and their expression in the potato plant (Solanum tuberosum L.)

The cDNA clones of two potato -tubulin genes were isolated from a tuberising stolon tip library. Analysis of 20 positive clones showed that they represented one or another of two different but very similar -tubulin genes, designated TUBST1 and TUBST2. The expression pattern of -tubulin genes in the potato plant was investigated by RNA blot analysis and by RT-PCR. Southern analysis of potato genomic DNA with coding and non-coding -tubulin probes revealed that there are multiple -tubulin genes in the potato genome and that there is likely to be considerable divergence in the 3 non-coding sequences. Phylogenetic analysis of plant -tubulin genes is described.