Mosaic Virus 35S RNA Promoter Research Articles

Characterization of the bark storage protein gene (JcBSP) family in the perennial woody plant Jatropha curcas and the function of JcBSP1 in Arabidopsis thaliana.

BackgroundBark storage protein (BSP) plays an important role in seasonal nitrogen cycling in perennial deciduous trees. However, there is no report on the function of BSP in the perennial woody oil plant Jatropha curcas.MethodsIn this study, we identified six members of JcBSP gene family in J. curcas genome. The patterns, seasonal changes, and responses to nitrogen treatment in gene expression of JcBSPs were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Overexpression of JcBSP1 in transgenic Arabidopsis thaliana was driven by a constitutive cauliflower mosaic virus (CaMV) 35S RNA promoter.ResultsJcBSP members were found to be expressed in various tissues, except seeds. The seasonal changes in the total protein concentration and JcBSP1 expression in the stems of J. curcas were positively correlated, as both increased in autumn and winter and decreased in spring and summer. In addition, the JcBSP1 expression in J. curcas seedlings treated with different concentrations of an NH4NO3 solution was positively correlated with the NH4NO3 concentration and application duration. Furthermore, JcBSP1 overexpression in Arabidopsis resulted in a phenotype of enlarged rosette leaves, flowers, and seeds, and significantly increased the seed weight and yield in transgenic plants.

Lettuce-based production of an oral vaccine against porcine edema disease for the seed lot system.

Plant-made oral vaccines can be a cost-effective method to control infectious diseases of humans and farm animals. Pig edema is a bacterial disease caused by enterohemorrhagic Escherichia coli producing the toxin Shiga toxin 2e (Stx2e). In our previous report, we chose the non-toxic B subunit of Stx2e (Stx2eB) as a vaccine antigen, and Stx2eB was expressed in lettuce (Lactuca sativa L., cv. Green wave). We found that a double repeated Stx2eB (2×Stx2eB) accumulates to higher levels than a single Stx2eB. In this study, we analyzed progeny plants introduced with 2×Stx2eB in which the gene was expressed under the control of conventional cauliflower mosaic virus 35S RNA (CaMV 35S) promoter, and found that the lettuce underwent transgene silencing and bore few seeds. We resolved these problems by using a transgene cassette which harbored a transcriptional promoter derived from the lettuce ubiquitin gene and a longer version of HSPT. The lettuce harboring this expression construct will be valuable in establishing the seed lot system on the basis that thousands of seeds can be obtained from one plant body and the resulting progeny plants accumulate 2×Stx2eB at high levels without the transgene silencing.

Obtainment and Analysis of Marker-Free Oil Plants Camelina sativa (L.) Expressing of Antimicrobial Peptide Cecropin P1 Gene

Marker-free transgenic Camelina sativa (L.) plants carrying a synthetic gene for cecropin P1, an antimicrobial peptide, under the control of the cauliflower mosaic virus 35S RNA promoter have been obtained and analyzed. The plants were transformed with an agrobacterial binary vector free of selective genes of antibiotic and herbicide resistance. The marker-free transformants were screened via measurement of the antibacterial activity of cecropin P1 and enzyme immunoassay. The obtained plants exhibited an increased resistance to infection with the bacteria Erwinia carotovora, the fungi Fusarium graminearum, and oxidative stress during infection. Analysis of the fatty acid composition of seed oil showed an increased amount of α-linolenic acid in the transgenic Camelina lines as compared to unmodified plants. The results indicate that the cecropin P1 gene can be included in an integral antistress plant-protective system.

Получение и анализ безмаркерных масличных растений Camelina sativa (L.), экспрессирующих ген антимикробного пептида цекропина Р1

The marker-free transgenic Camelina sativa (L.) plants carrying a synthetic gene for cecropin P1, an antimicrobial peptide, under the control of the cauliflower mosaic virus 35S RNA promoter have been obtained and analyzed. The plants were transformed by the agrobacterial binary vector free of selection genes of antibiotic and herbicide resistance. Screening of the marker-free transformants was performed by measuring the antibacterial activity of cecropin P1 and by enzyme immunoassay. The obtained plants exhibited increased resistance to the infection by the Erwinia carotovora bacteria, Fusarium graminearum fungi, and to oxidative stress during infection. The analysis of the fatty acid composition of seed oil showed an increased amount of α-linolenic acid in the transgenic camelina lines as compared to unmodified plants. The obtained results indicate that the gene for cecropin P1 can be included in the integral antistress plant protective system. Camelina sativa, cecropin Р1, agrobacterium-mediated transformation, marker-free transgenic plants, fatty acid compositions, microbial phytopathogenes, oxidative stress, resistance, cecropin P1. The work was financially supported by State job № 0101-2014-0046, № РК 01201352439 and with the partial financial support the Russian Fund for Basic Investigation, Grants № 18-08-00752, № 16-04-00623.

35S Promoter Methylation in Kanamycin-Resistant Kalanchoe (Kalanchoe pinnata L.) Plants Expressing the Antimicrobial Peptide Cecropin P1 Transgene.

Transgenic kalanchoe plants (Kalanchoe pinnata L.) expressing the antimicrobial peptide cecropin P1 gene (cecP1) under the control of the 35S cauliflower mosaic virus 35S RNA promoter and the selective neomycin phosphotransferase II (nptII) gene under the control of the nopaline synthase gene promoter were studied. The 35S promoter methylation and the cecropin P1 biosynthesis levels were compared in plants growing on media with and without kanamycin. The low level of active 35S promoter methylation further decreases upon cultivation on kanamycin-containing medium, while cecropin P1 synthesis increases.

Tolerance to Excess-Boron Conditions Acquired by Stabilization of a BOR1 Variant with Weak Polarity in Arabidopsis.

Boron (B) is a metalloid that is essential for plant growth but is toxic when present in excess. Arabidopsis BOR1 is a borate exporter, facilitating B translocation from root to shoot under limited-B conditions. BOR1 shows stele side polar localization in the plasma membrane of various root cells, presumably to support B translocation toward the stele. BOR1 is degraded under high-B supply through vacuolar sorting via ubiquitination at the K590 residue to prevent the accumulation of B to a toxic level in shoots. A previous study showed that overexpression of BOR1 under control of the cauliflower mosaic virus 35S RNA promoter improved the growth of Arabidopsis under limited-B conditions without affecting the growth under sufficient-to-excess-B conditions. In this study, we unexpectedly found that ubiquitous expression of a stabilized BOR1 variant improved tolerance to excess-B in Arabidopsis. We established transgenic plants expressing BOR1-GFP fused with hygromycin phosphotransferase (HPT) and BOR1(K590A)-GFP-HPT under control of the ubiquitin 10 promoter. BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT were expressed in various cell types in leaves and roots and showed weak polar localization in root tip cells. BOR1-GFP-HPT, but not BOR1(K590A)-GFP-HPT, was degraded through an endocytic pathway under high-B conditions. Transgenic plants with the stabilized variant BOR1(K590A)-GFP-HPT showed improved root and shoot growth under excess-B conditions. The concentration of B was greater in the shoots of plants with BOR1(K590A)-GFP-HPT or BOR1-GFP-HPT than in those of untransformed wild-type plants. These results suggest that BOR1(K590A)-GFP-HPT confers tolerance to excess-B by excluding B from the cytosol of shoot cells. Results from this study indicate the potential for engineering the trafficking properties of a transporter to produce plants that are tolerant to mineral stress.

A technical breakthrough close at hand: feasible approaches toward establishing a gene-targeting genetic transformation system in seaweeds

The lack of a genetic transformation system that allows integration of a foreign gene into the genome is a serious hindrance to progress in biological research on seaweeds. In contrast, physiological research in seaweeds has a long history of focus on the establishment of cell polarity and multicellularity (Brownlee and Bouget, 1998; Hable and Hart, 2010). Today, nuclear genomes have been recently sequenced for many species of seaweeds and there is now a significant accumulation of genomic information in the form of expressed sequence tag data in public and private databases for analyses (see Mikami, 2014). Although physiological and molecular biological approaches have the potential to allow genome-wide and comprehensive analyses of factors involved in seaweed development, the lack of a genetic transformation system hinders functional identification of genes encoding regulatory components of seaweed development. This is impeding the application of the molecular biological knowledge in industrial and economic activities related to seaweed. Because genetic manipulation systems are indispensable for the functional analysis of genes, it is necessary to develop reverse genetic methods for gene knock-out, knock-in, or replacement in a variety of seaweeds via either homologous recombination or random integration of foreign DNA with a marker to identify its integration site in the genome. To accelerate progress toward establishing genetic transformation methods, there are four important issues to be resolved: (1) creating expression constructs particularly suited to seaweed cells; (2) transferring the expression constructs into cells; (3) ensuring integration of expression constructs into the genome; and (4) selecting genetically transformed cells. It has already been confirmed that combining codon-optimized coding regions and endogenous strong promoters results in high-efficiency expression in seaweed cells (Mikami et al., 2011; Mikami and Uji, 2012; Mikami, 2013). Seaweed genomes generally have high GC content and thus coding sequences should be biased toward higher amounts of G and C. Moreover, the Cauliflower mosaic virus (CaMV) 35S RNA promoter that has been used for foreign gene expression in seaweeds prior to establishment of the transient gene expression has rather low activity in seaweed cells (Mikami et al., 2011; Mikami and Uji, 2012; Mikami, 2013). But importantly, neither the codon optimization nor endogenous promoters yet tested could enhance the expression of foreign genes in seaweeds (Mikami et al., 2011). These findings clearly indicate that codon-optimized coding regions directed by strong endogenous promoters will be required elements of the design of constructs that can be expressed efficiently in seaweed cells (Mikami et al., 2011; Mikami and Uji, 2012; Mikami, 2013). This strategy has already been applied to a wide variety of red seaweeds (Hirata et al., 2011a,b; Son et al., 2011). Moreover, the second requirement for genetic transformation, to transfer expression constructs into cells, has been resolved using particle bombardment technology in red seaweeds (Mikami et al., 2011; Mikami and Uji, 2012; Mikami, 2013) and microinjection in Ectocarpus siliculosus (Farnham et al., 2013). However, neither electroporation nor glass bead methods have yet been successful for transferring DNA constructs into seaweed cells. Our preliminary data indicated that the glass bead method and various gene transfer regents designed for mammalian cells were ineffective for cell wall–less protoplasts and monospores from the red seaweed Pyropia yezoensis. Taken together, the third and fourth issues—to ensure integration of expression constructs into the genome, and select genetically transformed cells, respectively—still have to be resolved to establish nuclear genetic transformation.

Improved tolerance to boron deficiency by enhanced expression of the boron transporter BOR2

Boron (B) cross-links the pectin polysaccharide rhamnogalacturonan II (RG-II) and thus is important for cell wall structure in plants. B deficiency is an agricultural problem that causes significant losses of crop productivity worldwide. To address this, B deficiency-tolerant plants have been generated using B transporters. With the goal of further improving plant tolerance to low-B conditions, we generated transgenic Arabidopsis thaliana (L.) Heynh. with enhanced expression of BOR2, a B transporter that promotes cross-linking of RG-II and root elongation under low B supply. We generated a DNA construct containing the cauliflower mosaic virus 35S RNA promoter, a native promoter of BOR2, a BOR2 gene and green fluorescent protein (GFP) (Pro35S-BOR2:BOR2-GFP), and obtained three independent transgenic lines with relatively high levels of BOR2-GFP expression. In the transgenic lines, BOR2-GFP was expressed mainly in the lateral root caps in the meristem zone and in the epidermal cells in the elongation zone, similar to its expression when driven only by its native promoter. In the Pro35S-BOR2:BOR2-GFP lines, BOR2-GFP was also expressed in various cells in the maturation zone of roots and epidermal cells of shoots, where the expression was hardly detectable in ProBOR2:BOR2-GFP lines. The transgenic lines were cultured under various B concentrations on solid media and it was found that root growth of three lines and shoot growth of two lines were enhanced compared to wild-type plants under low-B conditions. This finding established that enhanced expression of BOR2 leads to improved root growth under low-B conditions. We also examined growth of the transgenic lines under hydroponic conditions. One of the three lines showed better growth and fertility under a low-B condition, while the wild type did not set seeds under the same condition, suggesting the potential utility of BOR2 expression in agricultural applications.

Functional expression of an animal type-Na+-ATPase gene from a marine red seaweed Porphyra yezoensis increases salinity tolerance in rice plants

Despite lacking Na+-ATPase as a sodium pump in vascular plants, a gene encoding KPA (K+ P-type ATPase), a putative animal type-Na+/K+-ATPase, has been isolated from the marine red alga Porphyra yezoensis and designated PyKPA1. To characterize the properties of PyKPA1 and also to confirm its ability to confer salinity tolerance in land plants, transgenic rice plants were produced that expressed the full-length PyKPA1 cDNA under the control of cauliflower mosaic virus 35S RNA promoter. We observed transcriptional activation of the transgene, plasma membrane-localization of the gene product fused with green fluorescent protein in onion epidermal cells, and Na+-ATPase activity in the plasma membrane fraction from transgenic rice plants, indicating that PyKPA1 was functionally expressed in rice plants. Transgenic lines were examined in terms of growth in salinity stress conditions, resulting in protection from a decrease in biomass, although growth of control rice plants was repressed. These results demonstrate the utility of a red algal animal type-sodium pump for conferring salinity tolerance to land plants.

Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast

The systematic creation of defined cell wall modifications in the model plant Arabidopsis thaliana by expression of microbial hydrolases with known specific activities is a promising approach to examine the impacts of cell wall composition and structure on both plant fitness and cell wall recalcitrance. Moreover, this approach allows the direct evaluation in living plants of hydrolase specificity, which can differ from in vitro specificity. To express genes encoding microbial hydrolases in A. thaliana, and target the hydrolases to the apoplast compartment, we constructed an expression cassette composed of the Cauliflower Mosaic Virus 35S RNA promoter, the A. thaliana β-expansin signal peptide, and the fluorescent marker protein YFP. Using this construct we successfully introduced into Colombia-0 plants three Aspergillus nidulans hydrolases, β-xylosidase/α-arabinosidase, feruloyl esterase, acetylxylan esterase, and a Xanthomonas oryzae putative a-L: -arabinofuranosidase. Fusion with YFP permitted quick and easy screening of transformants, detection of apoplastic localization, and protein size confirmation. Compared to wild-type Col-0, all transgenic lines showed a significant increase in the corresponding hydrolytic activity in the apoplast and changes in cell wall composition. Examination of hydrolytic activity in the transgenic plants also showed, for the first time, that the X. oryzae gene indeed encoded an enzyme with α-L: -arabinofuranosidase activity. None of the transgenic plants showed a visible phenotype; however, the induced compositional changes increased the degradability of biomass from plants expressing feruloyl esterase and β-xylosidase/α-arabinosidase. Our results demonstrate the viability of creating a set of transgenic A. thaliana plants with modified cell walls to use as a toolset for investigation of how cell wall composition contributes to recalcitrance and affects plant fitness.

Transgenic Belarussian-bred potato plants expressing the genes for antimicrobial peptides of the cecropin-melittin type

Binary vectors for Agrobacterium-mediated transformation were constructed to express the genes for antimicrobial peptides (APs) of the cectropin-melittin type under the control of the cauliflower mosaic virus 35S RNA promoter in plants. It was shown with Escherichia coli and Agrobacterium tumefaciens cells that the cassettes could be cloned in pB1121-based vectors with deletion of the 3-D-glycuronidase gene only in the orientation opposite to that of the original vector. Transgenic potato plants were obtained using the Belarussian varieties Odyssey, Vetraz, and Scarb. Their cells expressed the MsrA1 or CEMA peptides of the cecropin-melittin type. The expression was shown to confer higher resistance to bacterial (Erwinia carotovora) infection and extremely high resistance to fungal (Phytophtora infestans and Alternarla solani) infections.

Use of the gene of antimicrobial peptide cecropin P1 for producing marker-free transgenic plants

The marker-free transgenic tobacco plants carrying a synthetic gene encoding the antimicrobial peptide cecropin P1 (cecP1) under the control of the cauliflower mosaic virus 35S RNA promoter were produced. The binary vector pBM, free of any selective genes of resistance to antibiotics or herbicides intended for selecting transgenic plants, was used for transformation. The transformants were screened on a nonselective medium by detecting cecropin P1 in plant cells according to the antibacterial activity of plant extracts and enzyme immunoassay. According to the two used methods, 2% of the analyzed regenerants were transformants. The resulting marker-free plants displayed a considerably increased resistance to microbial phytopathogens-the bacterium Erwinia carotovora and fungus Sclerotinia sclerotiorum. Thus, the gene cecP1 can be concurrently used as a target gene and a screening marker. The utility of cecP1 as a selective gene for direct selection of transformed plants is discussed.

Highly Boron Deficiency-Tolerant Plants Generated by Enhanced Expression of NIP5;1, a Boric Acid Channel

Boron (B) is an essential element for plants, and B deficiency is a worldwide agricultural problem. In B-deficient areas, B is often supplied as fertilizer, but excess B can be toxic to both plants and animals. Generation of B deficiency-tolerant plants could reduce B fertilizer use. Improved fertility under B-limiting conditions in Arabidopsis thaliana by overexpression of BOR1, a B transporter, has been reported, but the root growth was not improved by the BOR1 overexpression. In this study, we report that enhanced expression of NIP5;1, a boric acid channel for efficient B uptake, resulted in improved root elongation under B-limiting conditions in A. thaliana. An NIP5;1 activation tag line, which has a T-DNA insertion with enhancer sequences near the NIP5;1 gene, showed improved root elongation under B limitation. We generated a construct which mimics the tag line: the cauliflower mosaic virus 35S RNA promoter was inserted at 1,357 bp upstream of the NIP5;1 transcription initiation site. Introduction of this construct into the nip5;1-1 mutant and the BOR1 overexpresser resulted in enhanced expression of NIP5;1 and improved root elongation under low B supply. Furthermore, one of the transgenic lines exhibited improved fertility and short-term B uptake. Our results demonstrate successful improvement of B deficiency tolerance and the potential of enhancing expression of a mineral nutrient channel gene to improve growth under nutrient-limiting conditions.

Improved translation efficiency in chrysanthemum and torenia with a translational enhancer derived from the tobacco alcohol dehydrogenase gene

We investigated the efficiency of the 5� -untranslated region (UTR) of the tobacco alcohol dehydrogenase gene (NtADH-5� UTR) as a translational enhancer in chrysanthemum (Chrysanthemum morifolium) and torenia (Torenia fournieri). Three constructs were introduced: control, ADH� , cauliflower mosaic virus 35S RNA (CaMV 35S) promoter::b-glucuronidase (GUS); NtADH-5� UTR with a spacer, ADHS, CaMV 35S promoter::NtADH-5� UTR::25-bp spacer::GUS; and NtADH-5� UTR with no spacer, ADH� , CaMV 35S promoter::NtADH-5� UTR::GUS. The highest GUS activity in ADHS and ADHfor chrysanthemum was about 45 and 190 times, respectively, and for torenia was 12 and 22 times, respectively, than that for the ADHplants. NtADH-5� UTR enhanced translational efficiency in both species. With the lowest translational efficiency set to 1, the relative translational efficiencies were 1 to 15 (ADH� ), 47 to 568 (ADHS), and 267 to 1360 (ADH� ) for chrysanthemum, and 1 to 3 (ADH� ), 47 to 114 (ADHS), and 85 to 226 (ADH� ) for torenia. NtADH-5� UTR would facilitate practical breeding and fundamental genetic research in chrysanthemum and torenia.

Assessment of CaMV-mediated gene silencing and integration of CaMV into GM plants with a 35S RNA promoter

Four GM plant species (Arabidopsis thaliana, Brassica napus, Nicotiana benthamiana and N. tabacum), each expressing the gene encoding the jellyfish green fluorescent protein (GFP) regulated by the cauliflower mosaic virus (CaMV) 35S RNA promoter, were assessed for the extent of transgene silencing and viral genome integration following infection by CaMV. The first three species are systemic hosts of CaMV, while N. tabacum is only a local host for a few strains of CaMV. A generalized systemic silencing of the GFP transgene was not observed in a total of 100 plants of each species infected with CaMV, although some localized loss of GFP was observed in CaMV-infected N. benthamiana leaves, and some loss of fluorescence was observed in older leaves of uninfected as well as infected plants. Progeny seedlings obtained from the above infected plants also did not exhibit transgene silencing showing that virus infection did not affect the stability of the transgene. These progeny plants also did not show signs of virus infection, indicating that the presence of the CaMV 35S RNA promoter sequences in the plant genome did not potentiate seed transmission of the virus. Integration of infective CaMV into the CaMV 35S RNA promoter could not be detected in 944 samples taken from leaves of the above infected plant species or in 2912 samples taken from progeny seedlings. Based on a detection limit of one copy per 4000 haploid genomes, we conclude that if integration of virus does occur into the CaMV 35S RNA promoter, then it occurs at such a low frequency as to be insignificant.

Characterization of FaRB7, a near root-specific gene from strawberry (Fragariaxananassa Duch.) and promoter activity analysis in homologous and heterologous hosts

Many insect and fungal pathogens posing agronomically important threats specifically target the roots in strawberry. The use of a root-specific promoter to confer expression of resistance genes in a targeted manner has the potential appreciably to benefit the genetic improvement of commercial strawberry varieties. A novel gene, FaRB7, was isolated from strawberry (Fragariaxananassa Duch.) and found to contain motifs characteristic of tonoplast intrinsic proteins (TIPs). Phylogenetic analysis revealed that FaRB7 represents an RB7-type TIP. In strawberry, this gene is expressed predominantly in roots, with very low expression in petioles. A 2.843 kb region representing the FaRB7 gene upstream regulatory sequence was isolated and found to share a number of sequence motifs with the promoter of the Nicotiana tabacum TobRB7 root-specific RB7-type TIP. When cloned upstream of the gusA reporter gene and introduced into strawberry plants, the FaRB7 promoter was shown to direct strong, near root-specific expression with expression patterns very similar to that of the endogenous gene. Furthermore, the FaRB7 promoter was found to confer constitutive expression, comparable to that produced by the cauliflower mosaic virus (CaMV) 35S RNA promoter, in tobacco. Thus, the FaRB7 promoter may be used to achieve near-root-specific transgene expression in strawberry and also represents an alternative to the CaMV 35S promoter for producing constitutive foreign gene expression in heterologous hosts. The FaRB7 full-length genomic sequence and 5' upstream regulatory region have been submitted to the EMBL/GenBank database under accession number DQ178022.

Improvement of seed yields under boron‐limiting conditions through overexpression of BOR1, a boron transporter for xylem loading, in Arabidopsis thaliana

Soil fertilization is a common practice in modern agriculture, undertaken to prevent nutrient deficiency in crops. However, fertilization is costly and causes environmental pollution. The cultivation of plants that tolerate low nutrient supplies may circumvent this problem. Here, we report the generation of Arabidopsis thaliana plants that tolerate boron (B)-deficient conditions due to the overexpression of BOR1, an efflux B transporter that is required for efficient xylem loading of B. In several independently generated transgenic plants expressing BOR1 or BOR1-GFP under the control of the cauliflower mosaic virus 35S RNA promoter, root-to-shoot translocation of B was enhanced and shoot growth was greater under B-limiting conditions compared with wild-type plants. In addition, the transgenic plants showed increased translocation of B, especially to the shoot apex, and set seed normally under B-limiting conditions, under which wild-type plants failed to set seed. This study therefore reports plants that show improved seed yields compared with wild-type under nutrient-deficient conditions as a result of increased production of an essential mineral nutrient transporter.

Detection of Genetic Modification in Cured Tobacco Leaf: Proficiency Testing Using Polymerase Chain Reaction-Based Methods

The Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA; Paris, France) "Task Force Genetically Modified Tobacco-Detection Methods" investigated the performance of qualitative and quantitative methods based on the polymerase chain reaction (PCR) for the detection and quantitation of genetically modified (GM) tobacco. In the 4 successful rounds of proficiency testing, the cauliflower mosaic virus 35S RNA promoter (CaMV 35S) and the Agrobacterium tumefaciens nopaline synthase terminator (NOS) were selected as target sequences. Blind-coded reference materials containing from 0.1 to 5.0% and from 0.15 to 4% GM tobacco were used in 2 rounds of qualitative and quantitative PCR, respectively. Eighteen laboratories from 10 countries participated in this study. Considering all methods and 2 rounds, the different laboratories were able to detect GM tobacco at the 0.1% level in 46 out of 58 tests in qualitative assays. The results of the proficiency test indicate that both end point screening and real-time quantitative methods are suitable for the detection of genetically modified organisms in tobacco leaf samples having a GM content of 0.1% or higher. The CORESTA proficiency study represents a first step towards the interlaboratory evaluation of accuracy and precision of PCR-based GM tobacco detection, which may lead to the harmonization of analytical procedures and to the enhancement of comparability of testing results produced by different laboratories.

Posttranslational Regulation of Nitrate Reductase Strongly Affects the Levels of Free Amino Acids and Nitrate, whereas Transcriptional Regulation Has Only Minor Influence

Diurnal variations in nitrate reductase (NR) activity and nitrogen metabolites were examined in wild-type Nicotiana plumbaginifolia and transformants with various degrees of NR deregulation. In the C1 line, NR was only deregulated at the transcriptional level by placing the NR gene under the control of the cauliflower mosaic virus 35S RNA promoter. In the Del8 and S521D lines, NR was additionally deregulated at the posttranslational level either by a deletion mutation in the N-terminal domain or by a mutation of the regulatory phosphorylation site (serine-521). Posttranslational regulation was essential for pronounced diurnal variations in NR activity. Low nitrate content was related to deregulation of NR, whereas the level of total free amino acids was much higher in plants with fully deregulated NR. Abolishing transcriptional and posttranslational regulation (S521D plants) resulted in an increase of glutamine and asparagine by a factor of 9 and 14, respectively, compared with wild type, whereas abolishing transcriptional regulation (C1 plants) only resulted in increases of glutamine and asparagine by factors <2. Among the minor amino acids, isoleucine and threonine, in particular, showed enhanced levels in S521D. Nitrate uptake rates were the same in S521D and wild type as determined with (15)N feeding. Deregulation of NR appears to set the level of certain amino acids, whereas diurnal variations were still determined by light/dark. Generally, deregulation of NR at the transcriptional level did not have much influence on metabolite levels, but additional deregulation at the posttranslational level resulted in profound changes of nitrogen metabolite levels.

Enhanced seed phytosterol accumulation through expression of a modified HMG‐CoA reductase

The regulation of phytosterol biosynthesis in seeds is of interest to biotechnologists because of the efficacy of dietary phytosterols in reducing blood cholesterol in humans. Mevalonate synthesis via 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) is a key step in phytosterol biosynthesis. HMG-CoA reductase is inactivated by phosphorylation by SNF1-related protein kinase 1 (SnRK1). With the aim of increasing seed phytosterol levels, transgenic tobacco plants were produced expressing a full-length Arabidopsis (Arabidopsis thaliana) HMG-CoA reductase gene (HMG1) coding sequence, a modified HMG1 sequence encoding a protein lacking the target serine residue for phosphorylation by SnRK1, or a chimaeric sequence encoding the N-terminal domain of the Arabidopsis HMG1 enzyme fused with the catalytic domain of yeast HMG-CoA reductase, which lacks an SnRK1 target site. All three transgenes (35S-AtHMG1, 35S-AtHMG1m and 35S-AtScHMG1) were under the control of a cauliflower mosaic virus 35S RNA promoter. Levels of seed phytosterols were up to 2.44-fold higher in plants transformed with the 35S-AtHMG1m gene than in the wild-type, and were significantly higher than in plants expressing 35S-AtHMG1 or 35S-AtScHMG1. In contrast, levels of phytosterols in leaves of plants transformed with the 35S-AtHMG1m gene were unchanged, suggesting that regulation of HMG-CoA reductase by SnRK1 is an important factor in seeds but not in leaves. A total of 11 independent transgenic lines expressing 35S-AtHMG1m or 35S-AtScHMG1 also showed an altered flower phenotype, comprising a compact floret, prolonged flowering, short, pale petals, a protruding style, short stamens, late anther development, little or no pollen production, premature flower abscission and poor seed set. Because of this phenotype, the modified HMG-CoA reductase gene would have to be expressed seed specifically if it were to be engineered into a crop plant for biotechnological purposes.