Engineering of Tomato Glandular Trichomes for the Production of Specialized Metabolites.

Glandular trichomes are specialized tissues on the epidermis of many plant species. On tomato they synthesize, store, and emit a variety of metabolites such as terpenoids, which play a role in the interaction with insects. Glandular trichomes are excellent tissues for studying the biosynthesis of specialized plant metabolites and are especially suitable targets for metabolic engineering. Here we describe the strategy for engineering tomato glandular trichomes, first with a transient expression system to provide proof of trichome specificity of selected promoters. Using microparticle bombardment, the trichome specificity of a terpene-synthase promoter could be validated in a relatively fast way. Second, we describe a method for stable expression of genes of interest in trichomes. Trichome-specific expression of another terpene-synthase promoter driving the yellow-fluorescence protein-gene is presented. Finally, we describe a case of the overexpression of farnesyl diphosphate synthase (FPS), specifically in tomato glandular trichomes, providing an important precursor in the biosynthetic pathway of sesquiterpenoids. FPS was targeted to the plastid aiming to engineer sesquiterpenoid production, but interestingly leading to a loss of monoterpenoid production in the transgenic tomato trichomes. With this example we show that trichomes are amenable to engineering though, even with knowledge of a biochemical pathway, the result of such engineering can be unexpected.

Ameliorating effects of industrial sugar residue on the Jales gold mine spoil (NE Portugal) using Holcus lanatus and Phaseolus vulgaris as indicators.

Phytostabilisation of bare heavily contaminated substrate, such as abandoned mine sites, is considered a very appropriate technology in order to diminish erosion and dispersion of contaminants into the surroundings. In this short-term pot study, application of industrial sugar residue (ISR), a waste product of the sugar industry, proved to ameliorate spoils conditions for plant performance by elevating pH and immobilising several metals. Although arsenate concentrations were positively correlated to spoil pH and spoil treatment with ISR mobilised As, growth of both Phaseolus vulgaris and Holcus lanatus improved significantly after applications of 3.75 g ISR kg(-1) dry spoil. Nutrient uptake from the substrate, with the exception of potassium, was elevated by ISR. As a remediation technique ISR application could be effective although in As-contaminated sites application might be restricted to areas where leaching to (ground) water does not form a risk.

The role of phytochelatins in arsenic tolerance in the hyperaccumulator Pteris vittata.

• Pteris vittata was the first identified arsenic (As) hyperaccumulator. Here we investigated whether phytochelatins (PCs) are involved in the hypertolerance of arsenic by P. vittata. • P. vittata was exposed to 0-500 µm arsenate for 5 d, or to 50 µm arsenate for 0-7 d. In addition, l-buthionine-sulphoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase, was used in combination with different arsenate exposures. The relationships between As accumulation and the concentrations of PCs and glutathione (GSH) were examined. • PC synthesis was induced upon exposure to arsenate in P. vittata, with only PC2 detected in the plant. The As concentration correlated significantly with PC2 concentration in both roots and shoots, but not with GSH. The molar ratio of PC-SH to As was c. 0.09 and 0.03 for shoots and roots, respectively, suggesting that only a small proportion (1-3%) of the As in P. vittata can be complexed with PCs. In the presence of arsenate, addition of BSO decreased PC2 concentrations in roots and shoots by 89-96% and 30-33%, respectively. BSO alone was found to inhibit root growth of P. vittata markedly. • The results suggest that PCs play a limited role in the hypertolerance of As in P. vittata.

Human platelet antigen-2 and -3 genotyping by PCR-SSP.

Allele-specific PCR using sequence specific primers (PCR-SSP) is a simple and reliable technique to detect point mutations in genes. We have developed a PCR-SSP to enable the detection of a C-T mutation at position 482 of the GPIb gene and a T-G mutation at position 13,962 in exon 26 of the GPIIb gene. These point mutations are at the basis of the HPA alloantigens 2a, 2b and 3a, 3b respectively. One primer of each primer set has a 3' nucleotide complementary to the DNA sequence coding for one allele. PCR product is only produced when the corresponding DNA is present and thus the genotype is determined by the presence or absence of a band in agarose electrophoresis of PCR products. A second set of primers in the same reaction yields a product regardless of the HPA genotype to control the efficiency of the PCR amplification. The HPA-2 and -3 genotypes determined in this way were in strict concordance with those established by conventional genotyping using PCR followed by restriction enzyme digestion (PCR-ASRA). PCR-SSP is a rapid and reliable technique that can be used for the determination of alleles which code for platelet alloantigens.

Rapid Rh D genotyping by polymerase chain reaction-based amplification of DNA.

Rh (rhesus) D is the dominant antigen of the Rh blood group system. Recent advances in characterization of the nucleotide sequence of the cDNA(s) encoding the Rh D polypeptide allow the determination of the Rh D genotype at the DNA level. This can be of help in cases in which red blood cells are not available for phenotyping, eg, when in concerns a fetus. We have tested three independent DNA typing methods based on the polymerase chain reaction (PCR) for their suitability to determine the Rh D genotype. DNA derived from peripheral blood mononuclear cells from 234 Rh-phenotyped healthy donors (178 Rh D positive and 56 Rh D negative) was used in the PCR. The Rh D genotypes, as determined with a method based on the allele-specific amplification of the 3' noncoding region of the Rh D gene described by Bennett et al (N Engl J Med 329:607, 1993), were not concordant with the serologically established phenotypes in all cases. We have encountered 5 discrepant results, ie, 3 false-positive and 2 false-negative (a father and child). Rh D genotyping with the second method was performed by PCR amplification of exon 7 of the D gene with allele-specific primers. In all donors phenotyped as D positive tested so far (n = 178), the results of molecular genotyping with this method were concordant with the serologic results, whereas a false-positive result was obtained in one of the D-negative donors (also false-positive in the first method). Complete agreement was found between genotypes determined in the third method, based on a 600-bp deletion in intron 4 of the Rh D gene described by Arce et al (Blood 82:651, 1993), and serologically determined phenotypes. The Rh blood group system is complex, and unknown polymorphisms at the DNA level are expected to exist. Therefore, although genotypes determined by the method of Arce et al were in agreement with serotypes, it cannot yet be regarded as the golden standard. More experience with this or other methods is still needed.

Rh E/e genotyping by allele-specific primer amplification.

It has been shown that the Rhesus (Rh) blood group antigens are encoded by two homologous genes: the Rh D gene and the Rh CcEe gene. The Rh CcEe gene encodes different peptides: the Rh C, c, E, and e polypeptides. Only one nucleotide difference has been found between the alleles encoding the Rh E and the Rh e antigen polypeptides. It is a C-->G transition at nucleotide position 676, which leads to an amino acid substitution from proline to alanine in the Rh e-carrying polypeptide. Here we present an allele-specific primer amplification (ASPA) method to determine the Rh E and Rh e genotypes. In one polymerase chain reaction, the sense primer had a 3'-end nucleotide specific for the cytosine at position 676 of the Rh E allele. In another reaction, a sense primer was used with a 3'-end nucleotide specific for the guanine at position 676 of the Rh e allele and the Rh D gene, whereas the antisense primer had a 3'-end nucleotide specific for the adenine at position 787 of the Rh CcEe gene. We tested DNA samples from 158 normal donors (including non-Caucasian donors and donors with rare Rh phenotypes) in these assays. There was full concordance with the results of serologic Rh E/e phenotyping. Thus, we may conclude that the ASPA approach leads to a simple and reliable method to determine the Rh E/e genotype. This can be useful in Rh E/e genotyping of fetuses and/or in cases in which no red blood cells are available for serotyping. Moreover, our results confirm the proposed association between the cytosine/guanine polymorphism at position 676 and the Rh E/e phenotype.

Human platelet antigen-5 (Br) genotyping by ASPA: allele-specific primer amplification (PCR-SSP)

We have developed a PCR assay named ASPA (allele-specific primer amplification) to determine the HPA-5a and -5b genotypes. It consists of two PCR-reactions. One primer of each primer set has a 3'-end nucleotide which is specific for A or G at position 505 of the HPA-5b or -5a allele respectively. The HPA-5 genotypes determined in this way were strictly concordant with the genotypes established by the PCR-ASRA and with the phenotypes established using MAIPA. The ASPA is a rapid and reliable technique and can be used for the determination of alleles which code for platelet antigen allotypes.

Association of a variable number of tandem repeats (VNTR) in glycoprotein Ib alpha and HPA-2 alloantigens.

The human platelet alloantigen HPA-2(Koa/Kob) system is involved in two clinical syndromes, neonatal alloimmune thrombocytopenia and platelet transfusion refractoriness. We have previously described that the human platelet alloantigens HPA-2a(Kob) and HPA-2b(Koa), are caused by a Thr145Met amino acid polymorphism in the N-terminal globular domain of the human platelet glycoprotein (GP) Ib alpha. In the present study the question was addressed as to whether a genetic association exists between this Thr145Met polymorphism and the recently described variable number of tandem repeat (VNTR) polymorphism in GP Ib alpha. Such an association has already been suggested by serological analysis (Ishida et al., 1991). This VNTR polymorphism results from a 13-amino-acid sequence repeat in the macroglycopeptide region of GP Ib alpha. Therefore, we developed a PCR method to analyze the VNTR region of 106 normal individuals who were also analyzed for the HPA-2 polymorphism. In this method genomic DNA derived from mononuclear cells was purified, the polymorphic region was amplified by PCR and was electrophoresed on agarose gels. Differences in the size of the PCR products made VNTR typing possible. Genotyping for the HPA-2 system was done by allele-specific restriction site analysis of PCR products with the restriction enzyme Sfa NI. The DNA derived from 12 HPA-2(a-b+) subjects, contained only the B variant (with 3 repeats) of the VNTR polymorphism. The D variant (with 1 repeat) was only found in HPA-2a positive individuals. The C variant (with 2 repeats) was found to be strongly associated with HPA-2a. However, two members of a family with a HPA-2(a+b+) genotype were found to be homozygous for the C variant of the VNTR polymorphism. This shows that the C variant can also be associated with HPA-2b. The A variant (with 4 repeats) was not encountered in the population studied. The strong association of HPA-2 and VNTR polymorphism, lying 761 bp apart on the GP Ib alpha gene, indicates linkage disequilibrium.

Human platelet antigen-1 (Zw) typing of fetuses by analysis of polymerase chain reaction-amplified genomic DNA from amniocytes.

Prenatal typing for the human platelet antigens-1 (HPA) permits identification of a fetus at risk for neonatal alloimmune thrombocytopenia (NAITP) in cases of HPA-1 incompatibility in which the father is heterozygous for the HPA-1a antigen. Diagnostic cordocentesis and phenotyping of the fetal platelets are used for this purpose. We applied allele-specific restriction enzyme analysis on polymerase chain reaction (PCR)-amplified DNA purified from amniocytes. This assays allows early second trimester typing for HPA-1 alleles. We were able to determine the genotype of three fetuses at risk. Iatrogenic fetal loss is lower with amniocentesis than with cordocentesis. Therefore, this technique is a welcome addition to the antenatal management of NAITP.

Sequence analysis of cDNA derived from reticulocyte mRNAs coding for Rh polypeptides and demonstration of E/e and C/c polymorphisms.

RNA derived from enriched reticulocytes of Rh-phenotyped donors was isolated, reversely transcribed into cDNA and amplified with Rh-specific primers by polymerase chain reaction. Nucleotide sequence analysis of the entire coding region of the Rh cDNAs was carried out. Four types of cDNAs were identified, tentatively designated as RhSCI, RhSCII, RhSCIII and RhSCIV. Comparison of RhSCII with RhSCI (identical to the previously reported RhIXb/30A cDNA), showed single base pair difference. Since RhSCI and RhSCII were found to be related to the presence of E or e antigen, respectively, the P226A amino acid polymorphism appears to be the genetic basis of the E/e polymorphism. RhSCIII was demonstrated to be a transcript derived from the RhD gene, with 35 amino acid substitutions as compared to RhSCI. RhSCIV was found to be present only in RhC-positive individuals, indicating that RhSCIV encodes a polypeptide carrying the C antigen. Six nucleotide changes, resulting in four amino acid substitutions W16C, L60I, N68S and P103S, were observed between RhSCII and RhSCIV, probably representing the C/c polymorphism.

Determination of human platelet antigen frequencies in the Dutch population by immunophenotyping and DNA (allele-specific restriction enzyme) analysis.

Platelets from 200 random Dutch blood donors were typed for the human platelet alloantigens HPA-1 to -5 recognized at present and for Naka. Naka is an epitope on glycoprotein IV, not expressed on the platelet of individuals with hereditary GP IV deficiency. Platelet immunofluorescence and monoclonal antibody-specific immobilization of platelet antigens (MAIPA) were applied for this purpose. The observed phenotype frequencies were 97.86% and 28.64% for HPA-1a and -1b, 100% and 13.15% for HPA-2a and -2b, 80.95% and 69.84% for HPA-3a and -3b, 100% and 0% for HPA-4a and -4b, 100% and 19.7% for HPA-5a and HPA-5b, respectively. Platelets from all donors reacted with the anti-Naka antibodies. To determine the gene frequencies for the HPA-1, HPA-2 and HPA-3 systems directly, DNA from 98 of these donors was isolated from peripheral blood mononuclear leucocytes and specific fragments were amplified by polymerase chain reaction (PCR). The fragments were analyzed using allele-specific restriction enzymes (ASRA). In all amplified PCR products an "internal control" for each assay, ie, a restriction site for the applied enzyme independent from the phenotype of the donor was present. In all donors tested, phenotypes, as determined by serological methods and genotypes, directly determined by the ASRA, were identical. Thus, the PCR-ASRA described in this report is a practical and reliable technique for the determination of alleles that code for platelet antigen allotypes, at least in the Dutch population.

Localization of the platelet-specific HPA-2 (Ko) alloantigens on the N-terminal globular fragment of platelet glycoprotein Ib alpha.

The human platelet-specific alloantigens HPA-2a and HPA-2b (= Kob and Koa) together constitute a biallelic antigen system. The HPA-2 antigens have not, to date, been located on a particular platelet membrane molecule. Here, we describe the localization of these antigens on platelet glycoprotein (GP) Ib alpha. Platelets from two patients with the Bernard-Soulier syndrome (BSS) were HPA-2(a-,b-) in the immunofluorescence test with HPA-2 alloantibodies on chloroquine-treated platelets. With monoclonal antibody (MoAb) immobilization of platelet antigen assay (MAIPA), positive reactions were obtained only when MoAbs against the platelet GPIb/IX complex were used in combination with anti-HPA-2a or -2b alloantibodies and normal donor platelets. By immunoprecipitation under nonreducing and reducing conditions a protein of 160 Kd and 145 Kd, respectively, was precipitated by the anti-HPA-2a serum. A protein migrating identically to this was precipitated by anti-GPIb MoAb. Normal donor platelets became HPA-2(a-,b-) after elastase treatment, suggesting that anti-HPA-2 antibodies bind to the N-terminal elastase-sensitive part of GPIb alpha. Anti-HPA-2a antibodies inhibited the ristocetin-induced agglutination of HPA-2a-positive platelets but not of HPA-2a-negative platelets, indicating that the epitopes recognized by these alloantibodies are localized in the proximity of the von Willebrand-factor-binding domain. Together, these data provide evidence that the HPA-2 alloantigens are located on the N-terminal globular elastase-sensitive part of GPIb alpha. Furthermore, we show that the recently described Siba antigen is probably identical to HPA-2a.