Soybean seeds overexpressing asparaginase exhibit reduced nitrogen concentration.

In soybean seed, a correlation has been observed between the concentration of free asparagine at mid-maturation and protein concentration at maturity. In this study, a Phaseolus vulgaris K+ -dependent asparaginase cDNA, PvAspG2, was expressed in transgenic soybean under the control of the embryo specific promoter of the ß-subunit of ß-conglycinin. Three lines were isolated having high expression of the transgene at the transcript, protein and enzyme activity levels at mid-maturation, with a 20- to 40-fold higher asparaginase activity in embryo than a control line expressing ß-glucuronidase. Increased asparaginase activity was associated with a reduction in free asparagine levels as a percentage of total free amino acids, by 11-18%, and an increase in free aspartic acid levels, by 25-60%. Two of the lines had reduced nitrogen concentration in mature seed as determined by nitrogen analysis, by 9-13%. Their levels of extractible globulins were reduced by 11-30%. This was accompanied by an increase in oil concentration, by 5-8%. The lack of change in nitrogen concentration in the third transgenic line was correlated with an increase in free glutamic acid levels by approximately 40% at mid-maturation.

Coloring genetically modified soybean grains with anthocyanins by suppression of the proanthocyanidin genes ANR1 and ANR2.

Detection and quantification of the levels of adventitious presence of genetically modified (GM) soybeans in non-GM grain shipments currently requires sophisticated tests that can have issues with their reproducibility. We show here that pigment biosynthesis in the soybean seed coat can be manipulated to provide a distinct color that would enable the simple visible detection of the GM soybean grain. We observed that a distinct red-brown grain color could be engineered by the simultaneous suppression of two proanthocyanidin (PA) genes, ANTHOCYANIDIN REDUCTASE1 (ANR1) and ANR2. Multiple reaction monitoring by liquid chromatography tandem mass spectrometry was used to quantify differentially accumulated seed coat metabolites, and revealed the redirection of metabolic flux into the anthocyanin pigment pathway and unexpectedly the flavonol-3-O-glucoside pathway. The upregulations of anthocyanin isogenes (DFR1 and GST26) and the anthocyanin/flavonol-3-O-glycosyltransferase (UGT78K2) were identified by quantitative RT-PCR to be endogenous feedback and feedforward responses to overaccumulation of upstream flavonoid intermediates resulting from ANR1 and ANR2 suppressions. These results suggested the transcription of flavonoid genes to be a key component of the mechanism responsible for the redirection of metabolite flux. This report identifies the suppression of PA genes to be a novel approach for engineering pigmentation in soybean grains.

Transfection of Arabidopsis protoplasts with a Plum pox virus (PPV) infectious clone for studying early molecular events associated with PPV infection.

The development of novel strategies against plant viral diseases relies on a better understanding of molecular virus-host interactions. Here, we report an easy, efficient and reproducible protocol for Arabidopsis protoplast isolation and transfection to study the infection and replication of a potyvirus, Plum pox virus (PPV). Macerozyme and cellulose were used to release protoplasts from Arabidopsis leaf tissues, and polyethylene glycol-mediated DNA uptake was employed for transfection of a PPV infectious clone. Protoplast viability was monitored by fluorescein diacetate staining, and transfection efficiency was estimated by transient expression of the green fluorescent protein. The protocol allowed production of 95% viable mesophyll protoplasts and a successful transfection rate of 35%. The system was used further in a time-course experiment to investigate PPV viral RNA accumulation. It was found that 3 h post-transfection (hpt) in the transfected protoplasts viral RNA increased by about 150-fold and progressively accumulated to reach the maximum at 12 hpt. Viral RNA then decreased dramatically at 24 hpt reaching 40% of its peak level. Considering the availability of the whole genome microarrays, and other genomic resources of Arabidopsis, the synchronized single-cell (protoplast) infection system will be useful for elucidating early molecular events associated with PPV infection.

Analysis and use of the tobacco eIF4A-10 promoter elements for transgene expression.

The eIF4A gene family codes for proteins which unwind secondary structures of mRNA during translational initiation. The tobacco eIF4A-10 promoter is one of a few of constitutive promoters found in plants. Research was conducted to identify the proximal promoter elements and to evaluate the potential application of the promoter for regulating transgene expression in a range of crop plants. A large intron (892 bp) in the leader sequence was found to be dispensable for constitutive promoter activity and did not contribute to the overall performance of the promoter. Deletion analysis showed that the upstream region between -151 bp and -73bp relative to the transcriptional start site was essential for the high level of expression and the constitutive activity. The data indicated that the elements in this region may coordinate and compensate each other for the high levels of promoter expression. The downstream leader sequence also contained a strong quantitative enhancer element that was essential for the full activity of the eIF4A-10 promoter. The eIF-4A10 promoter was found to be active in a wide range of plant species and tissues indicating that it will be useful for the constitutive expression of transgenes in plants.

Seed orientation and magnetic field strength have more influence on tomato seed performance than relative humidity and duration of exposure to non-uniform static magnetic fields.

Different factors (e.g., light, humidity, and temperature) including exposure to static magnetic fields (SMFs), referred here as critical factors, can significantly affect horticultural seed performance. However, the link between magnetic field parameters and other interdependent factors affecting seed viability is unclear. The importance of these critical factors affecting tomato (Solanum lycopersicum L.) var. MST/32 seed performance was assessed after performing several treatments based on a L9 (3(4)) (four factors at three levels) orthogonal array (OA) design. The variable factors in the design were magnetic flux density (R1=332.1±37.8mT; R2=108.7±26.9mT; and R3=50.6±10.5mT), exposure time (1, 2, and 24h), seed orientation (North polarity, South polarity, and control - no magnetic field), and relative humidity (RH) (7.0, 25.5, and 75.5%). After seed moisture content stabilisation at the different chosen RH, seeds were exposed in dark under laboratory conditions to several treatments based on the OA design before performance evaluation. Treatments not employing magnetic field exposure were used as controls. Results indicate that electrolyte leakage rate was reduced by a factor of 1.62 times during seed imbibition when non-uniform SMFs were employed. Higher germination (∼11.0%) was observed in magnetically-exposed seeds than in non-exposed ones, although seedlings emerging from SMF treatments did not show a consistent increase in biomass accumulation. The respective influence of the four critical factors tested on seed performance was ranked (in decreasing order) as seed orientation to external magnetic fields, magnetic field strength, RH, and exposure time. This study suggests a significant effect of non-uniform SMFs on seed performance with respect to RH, and more pronounced effects are observed during seed imbibition rather than during later developmental stages.

Micropropagation of ornamental Prunus spp. and GF305 peach, a Prunus viral indicator.

A micropropagation approach was developed for nine ornamental Prunus species, P. americana, P. cistena, P. glandulosa, P. serrulata 'Kwanzan', P. laurocerasus, P. sargentii, P. tomentosa, P. triloba, P. virginiana 'Schubert', commercially important in North America, and GF305 peach, commonly used for Prunus virus indexing. The micropropagation cycle based on proliferation of vegetative tissues includes establishment of tissue culture through introduction of shoot meristems in vitro, shoot proliferation, root induction and plant acclimatization steps and can be completed in 5 months. A meristem sterilization protocol minimized bacterial and fungal contamination. Multiple shoot formation in ornamental Prunus was obtained through the use of 1 mg l(-1) 6-benzyladenine. For GF305 peach, alteration in the sugar composition, fructose instead of sucrose, and addition of 1 mg l(-1 )ferulic acid had a significant impact on the shoot proliferation rate and maintenance of long-term in vitro culture. Rooting and plant acclimatization conditions were improved using a two-step protocol with a 4-day root induction in indole-3-butiric acid (IBA)-containing media with consequent 3-week root elongation in IBA-free media. One-month incubation of rooted shoots in a vermiculite-based medium resulted in additional shoot and root growth and provided better acclimatization and plant recovery. The micropropagation approach can be used for maintenance of the clonal properties for Prunus spp. as well as a protocol to support meristem therapy against viral infection.

High efficiency plant production of North American ginseng via somatic embryogenesis from cotyledon explants.

An efficient in vitro protocol for plant production of North American ginseng has been established. The pretreatment of cotyledon explants with 1.0 M sucrose at 4 degrees C resulted in an improvement of embryo quality and, combined with a higher sucrose content (7%) in induction medium, improved the embryogenesis frequency from 40% to 75% and the number of embryos per explant from 10 to 21. The frequency of secondary embryogenesis from somatic embryo-derived tissues cultured on MS medium with 1.0 mg l(-1) 2, 4-D and 1.0 mg l(-1) NAA is up to 90%. Somatic embryos can further develop to maturity on SH medium supplemented with 1% activated charcoal and half of them can germinate. About 85% of the germinated embryos will convert into plants with well-developed taproot systems on 1/2 SH medium with 0.5% activated charcoal. The growth chamber and field establishment rates were 95.6 and 93.7%, respectively. The plants transplanted to growth chambers and field plots appear normal.

Functional analysis of tomato Pti4 in Arabidopsis.

Pti4 is a tomato (Lycopersicon esculentum) transcription factor that belongs to the ERF (ethylene-responsive element binding factor) family of proteins. It interacts with the Pto kinase in tomato, which confers resistance to the Pseudomonas syringae pv tomato pathogen that causes bacterial speck disease. To study the function of Pti4, transgenic Arabidopsis plants were generated that expressed tomato Pti4 driven by the strong constitutive promoters, cauliflower mosaic virus 35S and tCUP. Global gene expression analysis by Affimetric GeneChip indicated that expression of Pti4 in transgenic Arabidopsis plants induced the expression of GCC box-containing PR genes. We also demonstrated that Pti4 enhanced GCC box-mediated transcription of a reporter gene. The data suggests that tomato Pti4 could act as a transcriptional activator to regulate expression of GCC box-containing genes. Furthermore, we show that the expression of tomato Pti4 in transgenic Arabidopsis plants produced a phenotype similar to that seen in plants treated with ethylene, thus providing evidence that the Pti4 gene is involved in the regulation of a subset of ethylene-responsive genes containing the GCC box.