PROTEINS INVOLVED IN MEMBRANE TRANSPORT BETWEEN THE ER AND THE GOLGI APPARATUS: 21 PUTATIVE PLANT HOMOLOGUES REVEALED BY DBEST SEARCHING

Numerous proteins have been identified in yeast and mammalian cells which are involved in trafficking between the endoplasmic reticulum and the Golgi apparatus. A great number of partial cDNA sequences now available from the two major plant model species, Arabidopsis thaliana and Oryza sativa, makes it possible to identify putative plant homologues of known genes/proteins from non-plant species. The authors used this approach to screen the database of Expressed Sequence Tags (dbEST) in order to detect plant homologues of proteins involved in membrane transport between ER and Golgi. Availability of these partial sequences will facilitate the screening of cDNA and genomic libraries otherwise performed using heterologous probes derived from animal and yeast genes. As the plant Golgi complex differs in many respects from its mammalian and yeast counterparts, the dbEST clones found can be directly used for various functional assays (immunoprecipitation, two-hybrid analysis, transgenic plants etc.) to test the exact roles of the encoded proteins and identify their functional partners, some of which may be specific for plants.     

Plant cryopreservation: a continuing requirement for food and ecosystem security

This issue of In Vitro Cellular and Developmental Biology—Plant is dedicated to current developments in liquid-nitrogen cryopreservation methods and their use in plant biology and germplasm preservation. The development of cryopreservation for storage of plant cells, tissues, and organs began in the 1960s and continues to this day. Long-term storage of in vitro cultures of secondary metabolite cell cultures, embryogenic cultures, clonal germplasm, endangered species, and transgenic products remains an important requirement for many scientists, organizations, and companies. The continued development of cryopreservation techniques and their application to new plants is the subject of this issue.     

Plasma membrane display of anti-viral single chain Fv fragments confers resistance to tobacco mosaic virus

We tested the hypothesis that membrane-anchored anti-viral antibodies can confer viral resistance to transgenic plants. A heterologous expression system was developed for plasma membrane targeting of anti-viral antibodies using mammalian transmembrane domains. A tobacco mosaic virus (TMV) neutralizing single-chain Fv antibody fragment (scFv24) was targeted to the endoplasmic reticulum and integrated into the plasma membrane of tobacco cells, using mammalian signal peptides and membrane receptor transmembrane domains. The human platelet-derived growth factor receptor (PDGFR) transmembrane domain or the T-cell receptor -domain (TcR) transmembrane domain was fused to the C-terminus of TMV-specific scFv24 to target expression of scFv24 as an extracellularly facing plasma membrane protein. Western blot and ELISA analyses were carried out to confirm functional expression of the recombinant fusion proteins scFv24-PDGFR and scFv24-TcR in transgenic tobacco suspension cultures and transgenic plants. Immunofluorescence and electron microscopy showed that the TcR transmembrane domain targeted scFv24 to the tobacco plasma membrane. Bioassays of viral infection showed that transgenic tobacco plants expressing scFv24-TcR were resistant to TMV infection. These results demonstrated that membrane anchored anti-viral antibody fragments are functional, can be targeted to the plasma membrane in planta and are a novel approach for engineering disease-resistant crops.     

Screening of transgenic plants by amplification of unknown genomic DNA flanking T-DNA.

For the screening of transfer DNA (T-DNA) integration in transgenic plant material, we developed a method based on specific amplification of genomic plant DNA flanking T-DNA borders. This approach is possible because the length of the region flanking T-DNA extremity on a restriction fragment is specific to the integration locus. We have modified an adaptor ligation PCR technique developed for amplification of unknown DNA flanking known sequence. The PCR patterns obtained were specific and reproducible for different plants from a given transgenic line. Furthermore, the number of PCR products obtained could be considered a good estimation of the T-DNA copy number. When compared to Southern blot analysis, the PCR results give valuable complementary information about the complexity of the T-DNA integration pattern and also about the integrity of the T-DNA borders. We describe the applications of this approach to populations of transgenic Arabidopsis thaliana plants.     

An Assay for Express Screening of Potato Transformants by GFP Fluorescence

An express assay for screening of potato transformants by their GFP fluorescence intensities is developed. In comparison to the widely used methods of transgenic plant screening by PCR, Real-Time RTPCR or Northern-blotting, the GFP fluorescence assay needs no expensive reagents and takes less time. This approach may also be used for nondestructive screening of the T0 transgenic regenerants which can be further grown and used. To prove this assay reliability, the expression of the hGFP gene in the leaves of transgenic potato (cv. Skoroplodny) plants, determined by its mRNA accumulation, was compared to GFP fluorescence intensity in the micro-samples of aseptic plant leaves. The strong correlation between the results of these two methods is the evidence of positive dependence of GFP fluorescence intensity on the target mRNA content.     

On the ecological and evolutionary significance of storage in clonal plants

Environmental heterogeneity has received wide attention in clonal plant research over the last decade. Most studies have focussed on the effects of spatial variation in environmental conditions on the performance of ramets and genets, while the effects of temporal heterogeneity have only occasionally been studied. As a consequence, our current knowledge about functional responses of clonal plants to habitat patchiness is biased towards spatial aspects of environmental heterogeneity. Nevertheless, temporal changes in biotic and abiotic conditions do occur in most natural habitats, and they are very likely to affect plant growth and performance, and to create positive selection pressures on traits that can buffer plants against unfavorable consequences of this variability. Storage of resources is a widespread phenomenon in clonal plant species. Typical clonal structures such as stolons, rhizomes and hibernacles serve as storage organs. However, the ecologic significance of storage in clonal plant structures remains partly unclear. We suggest that the lack of understanding with respect to resource storage in clonal plants be related to our poor knowledge of ecologic implications of temporal habitat heterogeneity in natural environments. Resource storage can be understood as a safety measure against temporal changes in the growing conditions of plants. This paper summarizes existing information about the ecologic relevance of storage in clonal plants and it tries to develop a framework for further investigation of resource storage as a strategy to enhance the performance of clonal plants in temporally variable environments.     

Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants

Green fluorescent protein (GFP) has emerged as a powerful new tool in a variety of organisms. An engineered sGFP(S65T) sequence containing optimized codons of highly expressed eukaryotic proteins has provided up to 100-fold brighter fluorescence signals than the original jellyfish GFP sequence in plant and mammalian cells. It would be useful to establish a non-invasive, quantitative detection system which is optimized for S65T-type GFP, one of the brightest chromophore mutants among the various GFPs. We demonstrate here that highly fluorescent transgenic Arabidopsis can be generated, and the fluorescence intensity of whole plants can be measured under non-disruptive, sterile conditions using a quantitative fluorescent imaging system with blue laser excitation. Homozygous plants can be distinguished from heterozygous plants and fully fertile progenies can be obtained from the analyzed plants. In the case of cultured tobacco cells, GFP-positive cells can be quantitatively distinguished from non-transformed cells under non-selective conditions. This system will be useful in applications such as mutant screening, analysis of whole-body phenomena, including gene silencing and quantitative assessments of colonies from microorganisms to cultured eukaryotic cells. To facilitate the elucidation of protein targeting and organelle biogenesis in planta, we also generated transgenic Arabidopsis that stably express the plastid- or mitochondria-targeted sGFP(S65T). Etioplasts in dark-grown cotyledons and mitochondria in dry seed embryos could be visualized for the first time in transgenic Arabidopsis plants under normal growing conditions.     

琉璃苣BoD6D载体的构建及转化

运用植物基因工程手段构建琉璃苣BoD6D转化载体,为提高油料作物油份中γ-亚麻酸含量奠定基础.以琉璃苣基因组DNA为模板克隆BoD6D,构建酵母表达载体并转化酿酒酵母,对酵母进行诱导表达,提取脂肪酸后进行甲酯化反应,利用气相色谱分析脂肪酸含量;同时构建植物双元表达载体,经农杆菌介导通过蘸花法转化拟南芥,最后对转基因拟南...     

One gene member of the ADP-ribosylation factor family is heat-inducible and enhances seed germination in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

ADP ribosylation factors (ARFs), one group within the Ras superfamily of GTP-binding proteins, are ubiquitous within the eukaryotic kingdom. The functions of ARFs are extensive, and include regulatory roles in vesicular transportation, lipid metabolism, and microtubule dynamics, and the cellular processes related to these roles. Most ARFs have been identified from mammalian species and yeast; although little is known about the functional importance of ARFs in plants, it seems to be equally diverse and significant. We have been working on plant responses under heat stress, and showed that heat-shock can induce seed germination (Koo et al. in Plant Physiol 167:1030–1038, 2015). In the present study, we report nine ARF gene family members from tobacco (Nicotiana tabacum), all belonging to the same group (Class 1) in the phylogenetic analysis. One family member, NtARF1, was induced under high-temperature stress. To elucidate the biological function of NtARF1, we generated transgenic tobacco plants overexpressing NtARF1 and the seeds of these transgenic tobacco plants germinated earlier than the seeds of non-transgenic tobacco plants. We also classified ARF family genes in plant species through systematic genomic DNA sequence data-mining, focusing on the fully sequenced and extensively annotated genomes of Arabidopsis thaliana, Brachypodium distachyon, Medicago truncatula, Mimulus guttatus, Nicotiana benthamiana, Setaria italica, Solanum lycopercisum, and Solanum tuberosum, and of some major crops including rice, soybean, corn, and tobacco. The Class 1 of our phylogenetics analysis comprised the highest number of ARFs among the four groups obtained for all plant species analyzed, especially for crop plant species.     

Accurate identification of taxon-specific molecular markers in plants based on DNA signature sequence

Accurate identification of plants remains a significant challenge for taxonomists and is the basis for plant diversity conservation. Although DNA barcoding methods are commonly used for plant identification, these are limited by the low amplification success and low discriminative power of selected genomic regions. In this study, we developed a k-mer–based approach, the DNA signature sequence (DSS), to accurately identify plant taxon-specific markers, especially at the species level. DSS is a constant-length nucleotide sequence capable of identifying a taxon and distinguishing it from other taxa. In this study, we performed the first large-scale study of DSS markers in plants. DSS candidates of 3899 angiosperm plant species were calculated based on a chloroplast data set with 4356 assemblies. Using Sanger sequencing of PCR amplicons and high-throughput sequencing, DSSs were validated in four and 165 species, respectively. Based on this, the universality of the DSSs was over 79.38%. Several indicators influencing DSS marker identification and detection have also been evaluated, and common criteria for DSS application in plant identification have been proposed.     

Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures

The production of the blue dye indigo in plants has been assumed to be a possible route to the introduction of novel coloration into flowers or fibres. As the human cytochrome P450 mono-oxygenase 2A6 (CYP2A6) can form indigo in bacterial cultures, we investigated whether the expression of the corresponding cDNA in transgenic plants could lead to indigo formation. In a first attempt, we generated tobacco cell suspension cultures expressing the cDNA encoding human CYP2A6. Supplementation of the medium with indole led to the generation of indican (3-hydroxyindole-β- d -glucoside), a metabolite usually exclusively present in indigoferous dye plants. Hence, the recombinant CYP2A6 converted indole to the reactive metabolite 3-hydroxyindole (indoxyl), whereas rapid glucosylation is obviously conducted by ubiquitous plant glucosyl transferases (GTs). Interestingly, of nine additionally tested plant cell suspension cultures from various plant families, five were also capable of the formation of indican after indole supplementation, although this metabolism was more pronounced in transgenic tobacco cell suspension cultures expressing CYP2A6 cDNA. To evaluate whether indican or even indigo could be produced in whole plants, we generated transgenic tobacco plants harbouring active CYP2A6 together with an indole synthase (BX1) from maize. The genetically engineered tobacco plants accumulated indican, but did not develop a blue coloration. Although the de novo formation of indican in transgenic tobacco plants hampered indigo formation, it supports the contention that biosynthetic pathways can be efficiently mimicked by metabolic engineering.     

Recovery of Chimeric Rice Plants from Dry Seed using Electric Discharge Particle Acceleration

The development of an efficient system for the creation of transgenicrice plants from immature embryo tissue was described previously.That system resulted in the generation of clonal plants, probablyderived from a single cell or a very small number of cells.Creation of chimeric cereal plants is of interest in studiesdirected towards the elucidation of developmental pathways.We were able to take advantage of the relative ease with whichrice axillary buds, in a germinating seedling, can from embryogeniccallus or be induced to form multiple tillers. Particle bombardmentof such tissues, at various stages of development, resultedin the recovery of transgenic plants that were chimeric. Thecreation, origin and development of chimeric cereal plants canthus be studied and new insight into fundamental aspects ofearly plant development can be obtained. This regeneration/transformationsystem is compared with a previously developed protocol involvinggene transfer into immature embryos which resulted in the exclusiverecovery of clonal plants. It is also compared and contrastedwith a system described earlier for soybean engineering thatresulted on the recovery both chimeric and clonal plants.Copyright1995, 1999 Academic Press Oryza sativa, transformation, chimeric plant phenotypes, development, ß-glucuronidase, particle bombardment     

Rapid and accurate early-stage detection of T-DNA/plant flanking sequences of resistant kumquats

Agrobacterium-mediated genetic transformation is a widely applied tool in plant biotechnology. In this process, genes of interest are integrated into plant genomes via T-DNAs present on plasmids in Agrobacteria. Classical and standard methods for screening transformants, such as Southern blot, are inconvenient for most woodland plants because of extremely low transformation efficiency. For the purpose of identifying transgenic woody lines at early selection stages, a right-border T-DNA/plant conjunction sequence analysis was carried out. By analyzing these sequences, 15 out of 17 kanamycin-resistant kumquats were found to be integrated with foreign genes, and two or more copies were present in 33.3% of the transgenic lines, which is completely concordant with Southern blots. Moreover, T-DNA integration into plant nuclear DNA was random without any sequence hotspots, and cleavage sites are any base of the sequence ‘TGAC’. These results showed that this screening method could not only detect resistant woodland plants rapidly at the early selection stage, but unequivocally detect copy numbers. Compared with other screening technique, this method could save time and effort for conducting genetic transformation in woody plants, and also provides accurate integration information for transgenic plants.     

Signal transduction in maize and Arabidopsis mesophyll protoplasts.

Plant protoplasts show physiological perceptions and responses to hormones, metabolites, environmental cues, and pathogen-derived elicitors, similar to cell-autonomous responses in intact tissues and plants. The development of defined protoplast transient expression systems for high-throughput screening and systematic characterization of gene functions has greatly contributed to elucidating plant signal transduction pathways, in combination with genetic, genomic, and transgenic approaches.     

Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells.

To analyze genomic changes resulting from double-strand break (DSB) repair, transgenic tobacco plants were obtained that carried in their genome a restriction site of the rare cutting endonuclease I-SceI within a negative selectable marker gene. After induction of DSB repair via Agrobacterium-mediated transient expression of I-SceI, plant cells were selected that carried a loss-of-function phenotype of the marker. Surprisingly, in addition to deletions, in a number of cases repair was associated with the insertion of unique and repetitive genomic sequences into the break. Thus, DSB repair offers a mechanism for spreading different kinds of sequences into new chromosomal positions. This may have evolutionary consequences particularly for plants, as genomic alterations occurring in meristem cells can be transferred to the next generation. Moreover, transfer DNA (T-DNA), carrying the open reading frame of I-SceI, was found in several cases to be integrated into the transgenic I-SceI site. This indicates that DSB repair also represents a pathway for the integration of T-DNA into the plant genome.     

Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders

Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration.     

Variations in genomic DNA methylation during the long-term in vitro proliferation of oil palm embryogenic suspension cultures

Key message

The long-term proliferation of embryogenic cell suspensions of oil palm is associated with changes in both genomic methylation rates and embryogenic capacities.

Abstract

In the aim of exploring the relationship between epigenetic stability and the long-term in vitro proliferation of plant tissues, we have studied changes in genomic DNA methylation levels in embryogenic suspensions of oil palm (Elaeis guineensis Jacq.). Five embryogenic callus lines were obtained from selected hybrid seeds and then proliferated as suspension cultures. Each clonal line obtained from a single genotype was subdivided into three independent subclonal lines. Once established, cultures proliferated for 12 months and genomic DNA was sampled at 4 months intervals for the estimation of global DNA methylation rates through high performance liquid chromatography (HPLC) quantitation of deoxynucleosides. Our results show that in vitro proliferation induces DNA hypermethylation in a time-dependent fashion. Moreover, this trend is statistically significant in several clonal lines and shared between subclonal lines originating from the same genotype. Interestingly, the only clonal line undergoing loss of genomic methylation in the course of proliferation has been found unable to generate somatic embryos. We discuss the possible implications of genome-wide DNA methylation changes in proliferating cells with a view to the maintenance of genomic and epigenomic stability.     

Enhancement of innate immune system in monocot rice by transferring the dicotyledonous elongation factor Tu receptor EFR

The elongation factor Tu (EF-Tu) receptor (EFR) in cruciferous plants specifical y recognizes the N-terminal acetylated elf18 region of bacterial EF-Tu and thereby activates plant immunity. It has been...     

Specialized binary vector for plant transformation: expression of the Arabidopsis thaliana AHAS gene in Nicotiana tabacum.

We constructed a cosmid vector, pOCA18, designed for transferring plant genomic libraries from Agrobacterium tumefaciens to plants. Clones from a genomic library of Arabidopsis thaliana DNA in pOCA 18 were propagated stably in both Escherichia coli and A. tumefaciens. Clones from the pOCA18 A. thaliana library were used to construct transgenic Nicotiana tabacum plants; the DNA inserts were transferred intact in 10 out of 16 transgenic N. tabacum plants examined but were partially deleted in six others. Transgenic N. tabacum plants constructed with a mutant A. thaliana acetohydroxy acid synthase gene (from the pOCA18 library) that encodes an enzyme resistant to the herbicide chlorsulfuron were resistant to chlorsulfuron. A statistical analysis indicated that if the A. thaliana library contains 10(7) members and if 10(7) A. tumefaciens transconjugants containing the library were used to transform plant cells, then 2 x 10(4) transformed plant cells must be generated to have a 95% probability of constructing a transgenic plant carrying a specific DNA sequence from the A. thaliana library.     

Stable Expression of Adalimumab in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

Production of monoclonal antibodies and pharmaceutical proteins in transgenic plants has been the focus of many research efforts for close to 30 years. Use of plants as bioreactors reduces large-scale production costs and minimizes risk for human pathogens contamination. Stable nuclear transformation of the plant genome offers a clear advantage in agricultural protein production platforms, limited only by the number of hectares that can be cultivated. We report here, for the first time, successful and stable expression of adalimumab in transgenic Nicotiana tabacum plants. The plant-derived adalimumab proved fully active and was shown to rescue L929 cells from the in vitro lethal effect of rhTNFα just as effectively as commercially available CHO-derived adalimumab (Humira). These results indicate that agricultural biopharming is an efficient alternative to mammalian cell-based expression platforms for the large-scale production of recombinant antibodies.