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.

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.

Assessment, validation and deployment strategy of a two-barcode protocol for facile genotyping of duckweed species.

Lemnaceae, commonly called duckweeds, comprise a diverse group of floating aquatic plants that have previously been classified into 37 species based on morphological and physiological criteria. In addition to their unique evolutionary position among angiosperms and their applications in biomonitoring, the potential of duckweeds as a novel sustainable crop for fuel and feed has recently increased interest in the study of their biodiversity and systematics. However, due to their small size and abbreviated structure, accurate typing of duckweeds based on morphology can be challenging. In the past decade, attempts to employ molecular barcoding techniques for species assignment have produced promising results; however, they have yet to be codified into a simple and quantitative protocol. A study that compiles and compares the barcode sequences within all known species of this family would help to establish the fidelity and limits of this DNA-based approach. In this work, we compared the level of conservation between over 100 strains of duckweed for two intergenic barcode sequences derived from the plastid genome. By using over 300 sequences publicly available in the NCBI database, we determined the utility of each of these two barcodes for duckweed species identification. Through sequencing of these barcodes from additional accessions, 30 of the 37 known species of duckweed could be identified with varying levels of confidence using this approach. From our analyses using this reference dataset, we also confirmed two instances where mis-assignment of species has likely occurred. Potential strategies for further improving the scope of this technology are discussed.

Immunological assessment of plant-derived avian flu H5/HA1 variants.

Polypeptide variants of the HA1 antigenic domain of the H5N1 avian influenza virus hemagglutinin (HA) molecule were produced in plants using transient and stable expression systems and fused with His/c-myc tags or with mouse or human Fc antibody fragments. The resulting peptides were purified and used for intramuscular immunization of mice. While the recombinant HA1 variants induced a significant serum humoral immune response in the mice, none of the HA1 preparations induced virus-neutralizing antibodies. Fusion with the Fc fragment improved overall yield of the constructs and allowed purification requiring only a single step, but led to no detectable fusion-related enhancement of immunogenicity or quality of immune response.

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.

Comparison of nuclear ribosomal RNA genes among Solanum species and other Solanaceae.

The organization of the nuclear-encoded 18S, 5.8S, and 25S ribosomal RNA genes (ribosomal DNA; rDNA) of 21 New World species from different sections of the genus Solanum, of two Old World Solanum species, and of representatives of other Solanaceae (Nicotiana, Atropa, Datura, Physalis, and Capsicum) was analyzed by restriction enzyme mapping using different rDNA specific hybridization probes. All Solanum species investigated exhibited rDNA repeats between 8.7 and 9.3 kb in length; the only exception was S. neorossii with a repeat length of 10.3 kb. Sequence heterogeneity was observed mostly in the intergenic spacer (IGS) region. Restriction sites for EcoRI and DraI in the spacer sequences were found to be characteristic for the New World species of the genus Solanum and for Lycopersicon esculentum. An additional XbaI site was detected in the spacer region of two nontuber-bearing species, S. brevidens and S. etuberosum (subsection Estolonifera Hawkes; series Etuberosa), as well as in the primitive tuber-bearing species of the series Pinnatisecta and Polyadenia (subsection Potatoe G. Don), thus demonstrating that these Mexican species are separated from the other tuber-bearing species but are closely linked to the nontuber-bearing Estolonifera group. Two EcoRI sites mapped at the 3' end of the 25S rRNA coding region seem to be characteristic for members of the Solanaceae; the first EcoRI site is apparently methylated in approximately 50% of the rDNA repeats. Southern hybridization with an IGS fragment of Solanum tuberosum as hybridization probe and nucleotide sequence analysis of the phylogenetically informative 3' end of the 25S rDNA support the assumption that the New World species of the genus Solanum are closely related to Lycopersicon (tomato) in contrast with other Solanaceae investigated, Nicotiana, Atropa, Datura, Physalis, and Capsicum.(ABSTRACT TRUNCATED AT 250 WORDS)

Rhizosecretion of recombinant proteins from plant hairy roots.

Rhizosecretion of a target protein in the hydroponic medium provides an alternative manufacturing platform that simplifies the downstream purification procedure and increases protein yield. In order to increase the production rates of rhizosecreted proteins, we have exploited the ability of Agrobacterium rhizogenes to induce the formation of large amounts of root tissue on transgenic tobacco plants engineered to secrete a model recombinant protein, human secreted alkaline phosphatase (SEAP). The secretion of SEAP from hairy roots induced on the stems of transgenic tobacco plants was 5-7 times higher than that from adventitious transgenic roots.

Novel class of rDNA repeat units in somatic hybrids between Nicotiana and Atropa.

Behavior of ribosomal RNA genes in the process of somatic hybridization was analyzed using hybrids Nicotiana tabacum + Atropa belladonna. Blothybridization of parental species DNAs to (32)P-rDNA specific probes revealed two classes of ribosomal repeats in both tobacco and nightshade; their length was 11.2 kb, 10.4 kb (tobacco) and 9.4 kb, 10.2 kb (night-shade). For analysis of hybrids, labelled (32)P rDNA specific probes were hybridized to DNA of parental species and somatic hybrids digested with restriction endonucleases EcoR1, EcoRV and BamH1. A new class of ribosomal DNA repeat, absent in parental species, was found in hybrid line NtAb-1. Possible mechanisms of appearence of a new rDNA class in the process of somatic cell fusion are discussed.

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 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.

A quick and efficient system for antibiotic-free expression of heterologous genes in tobacco roots.

Requirement for antibiotic-resistance selection markers and difficulty in identifying transgenes with the highest expression levels remain the major obstacles for rapid production of recombinant proteins in plants. An alternative approach to producing transgenic plants free of antibiotic-resistance markers is the phenotypic-based selection with root-proliferation genes (rol genes) of Agrobacterium rhizogenes. By using Agrobacterium tumefaciens harboring the pRYG transformation vector with a cluster of rol genes linked to a heterologous gene of interest, we have developed a rapid transformation tool using hairy root formation as a selection marker. The expression of beta-glucuronidase in newly induced transgenic tobacco roots could be detected as early as 12 days after inoculation. Higher levels of transgene expression in the roots correlated positively with the rates of root elongation on hormone-free medium and thus could be used for positive selection. When tobacco plants were transformed with pRYG harboring the expression cassette for secreted alkaline phosphatase (SEAP), the release of SEAP from roots of the fully regenerated transgenic plants could be quantified at rates as high as 28 microg/g root dry weight per day.

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

Highly 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.

Calreticulin expression in plant cells: developmental regulation, tissue specificity and intracellular distribution.

The tissue-specific expression pattern and the intracellular distribution of the Ca(2+)-binding protein calreticulin at the mRNA and protein levels have been studied during somatic and zygotic embryogenesis of Nicotiana plumbaginifolia Viv. A full-length cDNA sequence encoding calreticulin was isolated from a lembda Zap cDNA library from early developmental stages of somatic embryogenesis. The deduced amino acid sequence of the calreticulin from N. plumbaginifolia shows high homology to the corresponding proteins of tobacco (98.2% identity), maize (80%) and barley (76.5%), and more than 55% homology to animal calreticulins, and the sequence motifs with established functions found in calreticulins of other species were quite conserved. Northern experiments revealed a developmental regulation of the calreticulin transcript with a maximum during the early stages of somatic embryogenesis and an auxin dependence during in-vitro cell culture. alpha-Naphthaleneacetic acid stimulated calreticulin expression whereas 2,4-dichlorophenoxyacetic acid reduced it. Immunohistological analysis of calreticulin distribution in the ovaries during zygotic embryogenesis showed that calreticulin biosynthesis started tissue specifically, with a high abundance in the endothelium of the integument in the ovules, followed by calreticulin accumulation in the embryo proper and in the associated endosperm at the late globular stage of embryogenesis. Using immunogold labeling, calreticulin was intracellularly localized with a high abundance to the Golgi compartment and to patches on the surface of dividing protoplasts. Smaller amounts were found in the endoplasmic reticulum and plasma membranes. The functional role of calreticulin in posttranslational processing and translocation processes, apart from its postulated function in cellular Ca2+ homeostasis, is discussed.

Elimination and rearrangement of parental rDNA in the allotetraploid Nicotiana tabacum.

Origin and rearrangement of ribosomal DNA repeats in natural allotetraploid Nicotiana tabacum are described. Comparative sequence analysis of the intergenic spacer (IGS) regions of Nicotiana tomentosiformis (the paternal diploid progenitor) and Nicotiana sylvestris (the maternal diploid progenitor) showed species-specific molecular features. These markers allowed us to trace the molecular evolution of parental rDNA in the allopolyploid genome of N. tabacum; at least the majority of tobacco rDNA repeats originated from N. tomentosiformis, which endured reconstruction of subrepeated regions in the IGS. We infer that after hybridization of the parental diploid species, rDNA with a longer IGS, donated by N. tomentosiformis, dominated over the rDNA with a shorter IGS from N. sylvestris; the latter was then eliminated from the allopolyploid genome. Thus, repeated sequences in allopolyploid genomes are targets for molecular rearrangement, demonstrating the dynamic nature of allopolyploid genomes.

Production of recombinant proteins in tobacco guttation fluid.

Guttation, the loss of water and dissolved materials from uninjured plant organs, is a common phenomenon in higher plants. By using endoplasmic reticulum signal peptides fused to the recombinant protein sequences, we have generated transgenic tobacco (Nicotiana tabacum L. cv Wisconsin) plants that secrete three heterologous proteins of different genetic backgrounds (bacterial xylanase, green fluorescent protein of jellyfish [Aequorea victoria], and human placental alkaline phosphatase) through the leaf intercellular space into tobacco guttation fluid. Production rates of 1.1 microg/g of leaf dry weight per day were achieved for alkaline phosphatase with this protein comprising almost 3% of total soluble protein in the guttation fluid. Guttation fluid can be collected throughout a plant's life, thus providing a continuous and nondestructive system for recombinant protein production. Guttation fluid has the potential of increasing the efficiency of recombinant protein production technology by increasing yield, abolishing extraction, and simplifying its downstream processing.

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.

Use of plant roots for phytoremediation and molecular farming.

Alternative agriculture, which expands the uses of plants well beyond food and fiber, is beginning to change plant biology. Two plant-based biotechnologies were recently developed that take advantage of the ability of plant roots to absorb or secrete various substances. They are (i) phytoextraction, the use of plants to remove pollutants from the environment and (ii) rhizosecretion, a subset of molecular farming, designed to produce and secrete valuable natural products and recombinant proteins from roots. Here we discuss recent advances in these technologies and assess their potential in soil remediation, drug discovery, and molecular farming.