Molecular cloning and sequence comparison of a cDNA encoding α-glucan, water dikinase (GWD) from potato (Solanum tuberosum L.), and analysis of gene expression

Starch phosphorylation is an important biochemical aspect of plant starch metabolism as it influences the overall structure of the starch granule, and a prerequisite for its degradation. There is a growing interest on the isolation and characterization of α-glucan/glucan-like, water dikinases (GWDs) from plants, particularly agriculturally important crops, because GWD is known to catalyze starch phosphorylation both in leaves and different plant storage organs. In the present study, a 4,789-bp full-length cDNA encoding a GWD isoform was isolated from a commercially important Indian potato cultivar, Kufri Chipsona-1 by RT-PCR approach using tuber RNA. The predicted protein consisted of 1,463 amino acids having N-terminal 77-amino acid transit peptide, and 1,386-amino acid mature protein shorter by one amino acid as compared to the other mature GWDs from potato and tomato. The mature GWD showed 98 % sequence identity with the GWD isolated earlier from the potato cv. Desiree. Variations were found at 25 locations representing mostly non-conservative substitutions. The GWD represents a distinct isoform from potato, as revealed by sequence and phylogenetic analyses. Amino acid composition, segment-wise hydrophobic characters, predicted secondary structures were also analyzed and documented in this report. Broadly, the level of GWD expression as analyzed by semi-quantitative RT-PCR approach was found to be nearly uniform both in the mature tubers and leaves from most of the potato cultivars. By immunodetection technique, a band corresponding to ~155 kDa protein was detected only in the tuber protein extracts. The tuber starch-bound phosphorus content data showed minor variations between the potato cultivars.     

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.     

Multiple elements of the S 2 -RNase promoter from potato (Solanum tuberosum L.) are required for cell type-specific expression in transgenic potato and tobacco

A functional analysis of the promoter of the S ₂ -RNase gene from potato was performed in transgenic potato and tobacco plants, using a deletion series of S ₂ -RNase promoter GUS fusions. A detailed histochemical and quantitative analysis of the transgenic tobacco plants revealed that S ₂ promoter fragments ranging in size from 5.6 kb in length down to 0.2 kb mediate a weak developmentally regulated expression in the pistil, and strong ectopic expression in pollen. In the pistil, different expression patterns were seen depending on the transformant, the predominant one being characterised by expression in the stigma and the transmitting tract of the style, whereas a few plants showed expression exclusively either in the stigma or in the stylar transmitting tissue. All transformants also showed GUS expression in the placental epidermis of the ovary. Two sequences that are conserved between the potato S ₁ -RNase and S ₂ -RNase promoters, termed motif I and motif III, are located in a fragment of the S ₂ promoter extending from position ?200 to bp ?100, and motif II, located between bp ?498 and ?480, was identified on the basis of sequence comparisons between pistil-specific promoters. Motif II was found to be dispensible for pistil-specific and for pollen-specific expression. Two submotifs, A and B, were identified within motif I. Both were essential for expression in the pistil but only B was necessary for expression in pollen. Although motif III has a similar bipartite structure and sequence to motif I, it was not sufficient to confer either pollen- or pistil-specific expression. However, deletion of motif III abolished pollen-specific expression in transient expression experiments, suggesting that an interaction between the two sequence motifs may be needed to specify cell type-specific expression. In transgenic potato the S ₂ -RNase promoter also mediates expression in pollen and in the pistil; however, significantly fewer plants showed expression than in tobacco, with most plants also exhibiting GUS expression in other tissues.     

Phenotypic variation and ploidy level of plants regenerated from protoplasts of tetraploid potato (Solanum tuberosum L. cv. ‘Bintje’)

Summary A wide range of phenotypic variation occurred among protoplast — derived plants of tetraploid potato cultivar Bintje. The variant plants had alterations in growth and vigour, and in leaf and stem characteristics. The results suggest that the altered morphologies are caused predominantly by changes in ploidy levels. Some alterations could be attributed typically to octoploidy and aneuploidy. The occurrence of mixoploidy indicates that at least part of the observed variation arose during culture stage. The exogeneous cytokinin or auxin level and their combination during in vitro phase influenced the frequency of the variants observed. The origin of variation is discussed.     

Genetic instability in protoclones of potato (Solanum tuberosum L. cv. ‘Bintje’): new types of variation after vegetative propagation

Summary The transmission of variation from protoplast-derived plants of tetraploid potato cultivar Bintje to tuber progeny was examined. The morphological alterations of a majority of the variant protoclones were transmitted to corresponding tuber progeny. Some of the normal and variant protoclones gave new phenotypes, or segregated into parental and new phenotypes after vegetative propagation. The ploidy levels of almost all these clones remained unchanged after propagation. It was concluded that the occurrence of variation after vegetative propagation was due to somatic segregation of chimeras resulting from gene mutations or chromosome structural rearrangements in only part of the regenerated plant. The origin of variation is discussed in the light of these results.     

The expression of petunia flavonoid 3′ and 3′5′ hydroxylase genes in potatoes (Solanum tuberosum cv. Jopung)

Flavonoid 3′ (F3′OH) and 3′5′ hydroxylase (F3′5′OH) play a major role in the synthesis of flavonoids. They are involved in the flavonoid modification and the B-ring hydroxylation produces quercetin and myricetin, respectively. We introduced the petunia F3′OH and F3′5′OH genes in potato and expression of these enzyme was confirmed by Southern and Northern blot analyses in these transgenic plants. In the flavonoid, staining experiment, all transgenic plants with petunia F3′OH and F3′5′OH genes were successfully changed with their green color to orange, confirming that quercetin was synthesized in those plants. Especially, the F3′5′OH transgenic potatoes showed the strongest orange color, and it was revealed by capillary electrophoresis that they produce quercetin one and a half times as much as the untransformed potatoes.     

Regulation of Agrobacterium tumefaciens T-cyt gene expression in leaves of transgenic potato (Solanum tuberosum L. cv. Désirée) is strongly influenced by plant culture conditions

The promoter region of the Agrobacterium tumefaciens T-cyt gene was linked in a translational fusion to the coding DNA of the reporter gene uidA (for -glucuronidase or GUS protein; EC 3.2.1.31) and to nos 3 flanking DNA. The chimaeric gene was introduced by Agrobacterium transformation into potato (Solanum tuberosum L. cv. Désirée). In nine transgenic lines, the average GUS levels were highest in extracts from stems and roots of in vitro grown plants (ca. 11 000 GUS activity units per pmol MU per mg protein per min) but lower in leaves of the in vitro grown plants (ca. 7000 units). GUS activity was intermediate in stems and roots of plants grown in soil as well as in in vitro crown galls (ca. 3000 units). Activity was low in tubers, irrespective of whether these developed in vitro or in soil (both ca. 100 units), and lowest of all in leaves of soil-grown plants (ca. 10–15 units). However, in shoot cultures reestablished from soil-grown plants, GUS activity in the leaves increased to that determined in the original shoot cultures. Hence, plant culture conditions strongly influenced the expression of the T-cyt-uidA-nos gene. In particular, it was silenced in leaves of soil-grown plants. The results are compared with previous analyses of the promoter region of the wild-type T-cyt gene and with the growth properties of a large number of crown gall cell lines and crown-gall-derived plants, including over forty S. tuberosum cv. Désirée cell lines isolated in the present study that were transformed with the wild-type T-cyt gene and six promoter-mutated derivatives. A number of implications are discussed for crown gall formation and for control of expression of plant genes which contain Activator or G-box type 5 expression control sequences.     

Amino acid differences between the α-chains from two hemoglobins of the yellow-cheeked vole (family cricetidae)

The yellow-cheeked vole (Microtus xanthognathus) shows two electrophoretic hemoglobin components. Electrophoresis of the polypeptide chains from the separated hemoglobin components shows identical β-chains but two α-chains of different mobility, α ^f and α ^s . The composition of soluble tryptic peptides was determined for each α-chain. Amino acid differences were found in peptides αT1 and αT9; the compositions of the remainder of the homologous peptides were identical. Differences in αT1, found at α4 (α ^s -Gly-α ^f -Val) and α5 (α ^s -Thr-α ^f -Asp), were confirmed after a run to residue 20 of the fast component in an automatic sequencer. The differences in charge between αT1 peptides can account for the electrophoretic pattern of two hemoglobins. This is the first time that it has been possible to identity the residues which can account for the charge difference between the two hemoglobins observed in a Microtus species.     

A group of α-1,4-glucan lyase genes from the fungi Morchella costata,M. vulgaris and Peziza ostracoderma. Cloning,complete sequencing and heterologous expression

We here report genes encoding a newly discovered class of starch- and glycogen-degrading enzyme, -1,4-glucan lyase (EC 4.2.2.13), which degrades starch and glycogen to 1,5-anhydro-D-fructose. Two lyases were purified and partially sequenced from the macrofungi Morchella costata and M. vulgaris. The obtained lyase amino acid sequences were used to generate PCR primers, which were further used to probe the fungal genomic libraries. Two lyase genes (Agll1;Mo.cos and Agll1;Mo.vul) from the two fungi were fully sequenced and found to contain a coding region of 3201 bp and 3213 bp, respectively. A total of 13 small introns were found in each of the two genes with identical positions. The two lyase genes share 86% identity at the amino acid level. They encode mature lyases with 1066 and 1070 amino acids, respectively. The deduced molecular masses of 121530 and 121971 Da agree with the values found for the two purified lyases. A structure analysis of the promoter regions of the lyase genes revealed a number of putative regulatory DNA elements, such as the AREA and CREA sites, which are related to nitrogen and carbon metabolism, respectively, and the CCAAT/CAAT boxes, which are related to basal expression of genes. A third lyase gene (Agll1;Pe.ost) from the fungus Peziza ostracoderma was partially sequenced to 557 bp. The amino acid sequence deduced from this nucleotide fragment shares 76% identity with the M. costata lyase. Heterologous expression of the M. costata lyase gene was achieved intracellularly in Pichia pastoris and Aspergillus niger.     

Characterization and expression analysis of P5CS (Δ1-pyrroline-5-carboxylate synthase) gene in two distinct populations of the Atlantic Forest native species Eugenia uniflora L.

Molecular Biology Reports - Eugenia uniflora is an Atlantic Forest native species, occurring in contrasting edaphoclimatic environments. The identification of genes involved in response to abiotic...     

Purification and characterization of two β-N-acetylhexosaminidases from the tobacco hornworm,Manduca sexta (L.) (Lepidoptera: Sphingidae)

β-N-Acetylhexosaminidases were detected in 10 insects including species of Lepidoptera, Coleoptera, Hemiptera, and Orthoptera. Two enzymes were purified from the tobacco hornworm, Manduca sexta (L.). EI was detected in larval and pharate pupal molting fluid, integument, and pupal hemolymph while EII was found in larval and pupal hemolymphs. They are acidic hydrolases with similar molecular weights (6.1 × 10⁴), molar extinction coefficients at 280 nm (1.9 × 10⁵ liters mol^?1 cm^?1), and pH optima (pH 6). They differ in the number of polypeptide chains per molecule (EI is a single chain and EII consists of two polypeptide chains), amino acid composition, extent of glycosylation (EII is probably a glycoprotein), isoelectric point (pI_EI = 5.9 and pI_EII ～- 5.1), tissue distribution, and reactivities toward nitrophenylated N-acetylglucosamine (k_cat,I = 328 s^?1 and k_cat,II = 103 s^?1) and N,N′-diacetylchitobiose (k_cat,I = 307 s^?1 and k_cat,II = 3 s^?1). These results suggest that EI is a chitinase and that EII may function as a hexosaminidase in vivo.     

Culture-independent analysis of an endophytic core microbiome in two species of wheat: Triticum aestivum L. (cv. ‘Hondia’) and the first report of microbiota in Triticum spelta L. (cv. ‘Rokosz’)

The main goal of the study was to determine the structure of endophytic bacteria inhabiting different parts (endosperm, germ, roots, coleoptiles, and leaves) of two wheat species, Triticum aestivum L. (cv. ‘Hondia’) and Triticum spelta L. (cv. ‘Rokosz’), in order to provide new knowledge about the stability and/or changeability of the core microbiome in different plant organs. The endophytic core microbiome is associated with plants throughout their whole life cycle; however, plant organs can determine the actual endophytic community. Therefore, next generation sequencing with MiSeq Illumina technology was applied to identify the endophytic microbiome of T. aestivum and T. spelta. Bioinformatic analyses were performed with the use of the DADA2(1.8) package and R software (3.5.1).It was demonstrated that wheat, which is an important crop plant, was associated with beneficial endophytic bacteria inside the endosperms, germs, roots, leaves, and coleoptiles. Importantly, for the first time, biodiversity was recognized in the coleoptiles of the investigated wheat species. Flavobacterium, Pseudomonas and Janthinobacterium were shown to be common genera for both tested wheat cultivars. Among them, Pseudomonas was found to be the only endophytic genus accompanying both wheat species from the endosperm stage to the development of the leaf. Paenibacillus was recognized as a core genus for the ‘Hondia’ cv., whereas Pedobacter and Duganella constituted the core microbiome in the ‘Rokosz’ cv. In addition, the first insight into the unique and yet unrecognized endophytic microbiome of T. spelta is presented.     

Clestobothrium neglectum (Lönnberg, 1893) n. comb. (Cestoda: Bothriocephalidae) from the tadpole fish Raniceps raninus (L.) (Gadidae) from Sweden

Clestobothrium neglectum (Lönnberg, 1893) n. comb. is redescribed from specimens previously deposited in The Natural History Museum, London, UK by Dr David I. Gibson obtained from the intestine of the tadpole fish Raniceps raninus (Gadidae) collected from off the western coast of Sweden, near Kristineberg. C. neglectum resembles C. crassiceps (Rudolphi, 1819), the type- and only species currently recognised in the genus, but differs in having fine spiniform-like structures that are likely microtriches covering the posterior fourth of the scolex and all proglottids, a highly folded tegument forming numerous longitudinal ridges on both the dorsal and ventral surfaces of the proglottids giving them a scalloped appearance, a somewhat smaller egg (68 × 35 vs 75 × 40 μm), a U-shaped rather than H-shaped ovary and more testes (70–85 vs 40–50 per proglottid). C. neglectum is also compared to two species with similar scoleces that were previously assigned to Clestobothrium, Bothriocephalus acheilognathi Yamaguti, 1934 and B. kivuensis Baer &; Fain, 1958 (considered to be synonymous with B. acheilognathi by Pool, 1987). This is the first report of a species of Clestobothrium Lühe, 1899 (Bothriocephalidae) from a gadid fish.