Nikolai Borisjuk

Plants and human health in the twenty-first century

The concept of growing crops for health rather than for food or fiber is slowly changing plant biotechnology and medicine. Rediscovery of the connection between plants and health is responsible for launching a new generation of botanical therapeutics that include plant-derived pharmaceuticals, multicomponent botanical drugs, dietary supplements, functional foods and plant-produced recombinant proteins. Many of these products will soon complement conventional pharmaceuticals in the treatment, prevention and diagnosis of diseases, while at the same time adding value to agriculture. Such complementation can be accelerated by developing better tools for the efficient exploration of diverse and mutually interacting arrays of phytochemicals and for the manipulation of the plants ability to synthesize natural products and complex proteins. This review discusses the history, future, scientific background and regulatory issues related to botanical therapeutics.

Production of recombinant proteins in plant root exudates.

The large-scale production of recombinant proteins in plants is limited by relatively low yields and difficulties in extraction and purification. These problems were addressed by engineering tobacco plants to continuously secrete recombinant proteins from their roots into a simple hydroponic medium. Three heterologous proteins of diverse origins (green fluorescent protein of jellyfish, human placental alkaline phosphatase [SEAP], and bacterial xylanase) were produced using the root secretion method (rhizosecretion). Protein secretion was dependent on the presence of the endoplasmic reticulum signal peptide fused to the recombinant protein sequence. All three secreted proteins retained their biological activity and, as shown for SEAP, accumulated in much higher amounts in the medium than in the root tissue.

Tobacco ribosomal DNA spacer element stimulates amplification and expression of heterologous genes

Here we show that the cis-acting genetic element aps (amplification-promoting sequence), isolated from the nontranscribed spacer region of tobacco ribosomal DNA (rDNA), increases the level of expression of recombinant proteins. Transgenic tobacco plants, transformed with expression cassettes containing the herbicide-resistant acetolactate synthase (hr-ALS) gene or the green fluorescent protein (GFP) gene fused to the aps sequence, had greater levels of corresponding messenger RNAs (mRNAs) and proteins compared to transformants lacking aps. Analysis of transgenic plants showed that aps increased the copy number and transcription of the adjacent heterologous genes and, in the case of hr-ALS, enhanced the herbicide resistance phenotype. Both the increased transgene copy number and enhanced expression were stably inherited. These data provide the first evidence that the aps sequence can be used for gene amplification in transgenic plants and possibly other multicellular organisms.

Structural analysis of rDNA in the genus Nicotiana

The nucleotide sequence of the intergenic spacer (IGS) region between the 25S and the 18S rRNA coding regions has been determined for tobacco (Nicotiana tabacum). The IGS (5140 bp in length) can be subdivided into several regions (I–VII) two of which, upstream and downstream of the putative transcription initiation site (TIS), contain prominent subrepeats (A and C). The unique sequence in the central part of the IGS (region IV) preceding the TIS is extremely AT-rich. The distance from the putative TIS to the 5′ end of the 18S rRNA gene is 3005 bp. The IGS sequences are compared with potato (Solanum tuberosum) and tomato (Lycopersicon esculentum) IGS. Restriction mapping of 13 Nicotiana species shows that considerable rDNA repeat length heterogeneity in this genus is probably due to different numbers of A and C subrepeats.

Nucleotide sequence of the potato rDNA intergenic spacer

The large intergenic spacer (IGS) of potato rDNA was sequenced and compared to the IGS sequence of tomato. Both spacers exhibit similar length and architecture. Absence of repeated elements down-stream of the putative transcription initiation site (TIS) in potato is compensated by the larger number of subrepeats upstream of the TIS. Especially high level of similarity (86% and 86.5%, respectively) is found in the AT-rich domain containing the TIS and the region approx. 800 bp upstream of the 18S rRNA gene.

Transcriptional regulation of cuticle biosynthesis

Plant cuticle is the hydrophobic protection layer that covers aerial plant organs and plays a pivotal role during plant development and interactions of plants with the environment. The mechanical structure and chemical composition of cuticle lipids and other secondary metabolites vary considerably between plant species, and in response to environmental stimuli and stresses. As the cuticle plays an important role in responses of plants to major abiotic stresses such as drought and high salinity, close attention has been paid to molecular processes underlying the stress-induced biosynthesis of cuticle components. This review addresses the genetic networks responsible for cuticle formation and in particular highlights the role of transcription factors that regulate cuticle formation in response to abiotic stresses.

A specific oligonucleotide of the 5S rDNA spacer and species-specific elements identify symmetric somatic hybrids between Solanum tuberosum and S. pinnatisectum

The nucleotide sequences of the 5S rRNA genes (5S rDNA) of two Solanum tuberosum breeding lines (R1 and B15) and of the Mexican wild species S. pinnatisectum were determined and compared with each other and to the 5S rDNA of other Solanaceae species (Lycopersicon esculentum, Nicotiana rustica and Petunia hybrida). The 5S rDNA repeats of the Solanum species are 324–329 bp in length, and they exhibit 91–95% sequence identity. Sequence variability is mainly located in a short region of the spacer separating the 5S rRNA coding regions. A synthetic 28-mer oligonucleotide constructed according to this region can be used as a specific hybridization probe to distinguish symmetric somatic hybrids between S. tubersosum breeding line B15 and S. pinnatisectum produced by protoplast fusion. Interestingly, the two Solanum breeding lines R1 and B15 differ also in this spacer region.

Distribution of novel and known repeated elements of Solanum and application for the identification of somatic hybrids among Solanum species

Species-specific repetitive DNA probes are a useful tool for the molecular identification of somatic hybrids. Therefore, the distribution of three repetitive DNA elements of Solanum was investigated in Solanum wild species, Solanum breeding lines, and in more distantly related species of the genera Lycopersicon, Nicotiana, and Datura. The clone pSCH15, obtained from S. circaeifolium, represents a new 168-bp repetitive element; it shows 73–79% sequence similarity to repetitive elements of S. brevidens and Lycopersicon species. The 163-bp element in pSBH6, cloned from S. bulbocastanum, turned out to be very similar (95% sequence homology) to the Lycopersicon element pLEG15/TGRI previously regarded to be present only in species of the genus Lycopersicon and in S. lycopersicoides. Lower sequence similarity of approximately 80% was observed to repetitive elements of S. brevidens which are organized differently. The repeats exhibited different degrees of specificity: by Southern hybridization the element represented by the clone pSBH6 could be detected in almost all Solanum species investigated here but only after long exposure to X-ray film. The previously described “Solanum-specific” element represented by the clone pSA287 was also found, although in a very low copy number, in Lycopersicon esculentum. Therefore, detection of the repetitive elements pSA287 and pSBH6 in those species in which the respective repeat is less represented depends on exposure time. In contrast, the element pSCH15 is prominently present only in a small number of Solanum wild species and — to some extent — in the diploid breeding lines as revealed after long exposure. Use of these repeated elements for the identification of specific genomes in protoplast-fusion hybrids between Solanum wild species and Solanum breeding lines, or between two breeding lines, was evaluated.

A Noninvasive Platform for Imaging and Quantifying Oil Storage in Submillimeter Tobacco Seed

Summary: This study describes a non-invasive NMR approach that enables counting of seeds inside the intact tobacco capsule, to measure seed sizes, to model the seed interior in three dimensions, to quantify the lipid content, and to visualize lipid gradients. While often thought of as a smoking drug, tobacco (Nicotiana spp.) is now considered as a plant of choice for molecular farming and biofuel production. Here, we describe a noninvasive means of deriving both the distribution of lipid and the microtopology of the submillimeter tobacco seed, founded on nuclear magnetic resonance (NMR) technology. Our platform enables counting of seeds inside the intact tobacco capsule to measure seed sizes, to model the seed interior in three dimensions, to quantify the lipid content, and to visualize lipid gradients. Hundreds of seeds can be simultaneously imaged at an isotropic resolution of 25 µm, sufficient to assess each individual seed. The relative contributions of the embryo and the endosperm to both seed size and total lipid content could be assessed. The extension of the platform to a range of wild and cultivated Nicotiana species demonstrated certain evolutionary trends in both seed topology and pattern of lipid storage. The NMR analysis of transgenic tobacco plants with seed-specific ectopic expression of the plastidial phosphoenolpyruvate/phosphate translocator, displayed a trade off between seed size and oil concentration. The NMR-based assay of seed lipid content and topology has a number of potential applications, in particular providing a means to test and optimize transgenic strategies aimed at the manipulation of seed size, seed number, and lipid content in tobacco and other species with submillimeter seeds.

Molecular analysis of highly repeated genome fractions in Solanum and their use as markers for the characterization of species and cultivars

SummaryHighly repeated DNA of potato (Solanum sp.) was characterized by cloning various major repeated elements of the nuclear genome. The percentage of the nuclear genome of the specific fractions and the restriction enzyme patterns were determined in order to show the distribution and organization of the respective repeats in the genome of Solanum tuberosum cultivars, dihaploid breeding lines and in wild species of Solanum. Several of the clones obtained were represented in a high copy number but showed no informative RFLP patterns. More information was gained from ‘restriction satellite’ repeats. The clone pR1T320 was found to contain satellite repeats (360 bp in length) that are proportionally present in the genome of all Solanum species at frequencies, between 0.5% and 2.6% and which are differently organized. This repeat was also found in the genera Lycopersicon, Datura and Nicotiana. With various restriction enzymes characteristic RFLP patterns were detected. A more or less genus-specific element for Solanum was the 183-bp repeat (clone pSA287; between 0.2–0.4% of the nuclear genome) that was present in the majority of the Solanum species analyzed except S. kurtzianum, S. bulbocastanum and S. pinnatisectum. In a few wild species (prominently in S. kurtzianum, S. demissum and S. acaule) a specific repeat type was detected (clone pSDT382; repeat length approximately 370 bp) that could be used to trace the wild species introduced into S. tuberosum cultivars. The repeats analyzed together with the 18S, 5.8S and 25S ribosomal DNA (1.9–5.2%, corresponding to 1800-5500 rDNA copies) comprised approximately 4–7% of the Solanum genome.