T1 and T2* mapping of the human quadriceps and patellar tendons using ultra-short echo-time (UTE) imaging and bivariate relaxation parameter-based volumetric visualization.

Quantification of magnetic resonance (MR)-based relaxation parameters of tendons and ligaments is challenging due to their very short transverse relaxation times, requiring application of ultra-short echo-time (UTE) imaging sequences. We quantify both T1 and T2* in the quadriceps and patellar tendons of healthy volunteers at a field strength of 3 T and visualize the results based on 3D segmentation by using bivariate histogram analysis. We applied a 3D ultra-short echo-time imaging sequence with either variable repetition times (VTR) or variable flip angles (VFA) for T1 quantification in combination with multi-echo acquisition for extracting T2*. The values of both relaxation parameters were subsequently binned for bivariate histogram analysis and corresponding cluster identification, which were subsequently visualized. Based on manually-drawn regions of interest in the tendons on the relaxation parameter maps, T1 and T2* boundaries were selected in the bivariate histogram to segment the quadriceps and patellar tendons and visualize the relaxation times by 3D volumetric rendering. Segmentation of bone marrow, fat, muscle and tendons was successfully performed based on the bivariate histogram analysis. Based on the segmentation results mean T2* relaxation times, over the entire tendon volumes averaged over all subjects, were 1.8 ms ±â€¯0.1 ms and 1.4 ms ±â€¯0.2 ms for the patellar and quadriceps tendons, respectively. The mean T1 value of the patellar tendon, averaged over all subjects, was 527 ms ±â€¯42 ms and 476 ms ±â€¯40 ms for the VFA and VTR acquisitions, respectively. The quadriceps tendon had higher mean T1 values of 662 ms ±â€¯97 ms (VFA method) and 637 ms ±â€¯40 ms (VTR method) compared to the patellar tendon. 3D volumetric visualization of the relaxation times revealed that T1 values are not constant over the volume of both tendons, but vary locally. This work provided additional data to build upon the scarce literature available on relaxation times in the quadriceps and patellar tendons. We were able to segment both tendons and to visualize the relaxation parameter distributions over the entire tendon volumes.

Relative and absolute test-retest reliabilities of biomechanical risk factors for knee osteoarthritis progression: benchmarks for meaningful change.

OBJECTIVE: Biomechanical factors are important treatment targets in knee osteoarthritis. The knee adduction (KAM) and flexion (KFM) moments, quadriceps strength and power, load frequency, and body mass index (BMI) all have the potential to affect knee articular cartilage integrity by modulating forces across the joint. To identify clinically meaningful change, however, these measurements must be reliable and sensitive to change. This study estimated relative and absolute test-retest reliabilities over long periods of biomechanical risk factors for knee osteoarthritis progression. METHOD: Data from a longitudinal, observational study were analyzed for knee osteoarthritis patients with data at baseline, 6-month and 24-month follow-ups. Gait kinematics and kinetics, quadriceps strength and power, daily load frequency and BMI were collected. Relative and absolute test-retest reliabilities of these measures were estimated using intraclass correlation coefficients (ICCs) and standard errors of measurement (SEMs), respectively. Minimal detectable change at the 95% confidence level (MDC95) was also calculated. RESULTS: Data from 46 participants [36 women; age 61.0 (6.6) years] were included. Good-to-excellent relative reliabilities (ICC ≥ 0.80) indicated that KAM peak and impulse, quadriceps strength and power, and BMI had a strong ability to discriminate amongst participants. Absolute reliabilities were high for quadriceps strength and BMI, which demonstrated reasonable within-participant variability (SEMs ≤ 11% of the mean). The MDC95 values supported use of clinical interventions effective in reducing BMI and KAM, and increasing quadriceps strength. CONCLUSION: These data are useful in interpreting findings from interventional or longitudinal investigations by determining whether observed changes are beyond measurement error and interpretable as true change.

Changes in time of sowing, flowering and maturity of cereals in Europe under climate change.

The phenological development of cereal crops from emergence through flowering to maturity is largely controlled by temperature, but also affected by day length and potential physiological stresses. Responses may vary between species and varieties. Climate change will affect the timing of cereal crop development, but exact changes will also depend on changes in varieties as affected by plant breeding and variety choices. This study aimed to assess changes in timing of major phenological stages of cereal crops in Northern and Central Europe under climate change. Records on dates of sowing, flowering, and maturity of wheat, oats and maize were collected from field experiments conducted during the period 1985-2009. Data for spring wheat and spring oats covered latitudes from 46 to 64°N, winter wheat from 46 to 61°N, and maize from 47 to 58°N. The number of observations (site-year-variety combinations) varied with phenological phase, but exceeded 2190, 227, 2076 and 1506 for winter wheat, spring wheat, spring oats and maize, respectively. The data were used to fit simple crop development models, assuming that the duration of the period until flowering depends on temperature and day length for wheat and oats, and on temperature for maize, and that the duration of the period from flowering to maturity in all species depends on temperature only. Species-specific base temperatures were used. Sowing date of spring cereals was estimated using a threshold temperature for the mean air temperature during 10 days prior to sowing. The mean estimated temperature thresholds for sowing were 6.1, 7.1 and 10.1°C for oats, wheat and maize, respectively. For spring oats and wheat the temperature threshold increased with latitude. The effective temperature sums required for both flowering and maturity increased with increasing mean annual temperature of the location, indicating that varieties are well adapted to given conditions. The responses of wheat and oats were largest for the period from flowering to maturity. Changes in timing of cereal phenology by 2040 were assessed for two climate model projections according to the observed dependencies on temperature and day length. The results showed advancements of sowing date of spring cereals by 1-3 weeks depending on climate model and region within Europe. The changes were largest in Northern Europe. Timing of flowering and maturity were projected to advance by 1-3 weeks. The changes were largest for grain maize and smallest for winter wheat, and they were generally largest in the western and northern part of the domain. There were considerable differences in predicted timing of sowing, flowering and maturity between the two climate model projections applied.

Long term prospective of the Seine River system: confronting climatic and direct anthropogenic changes.

To explore the evolution of a human impacted river, the Seine (France), over the 21st century, three driving factors were examined: climate, agriculture, and point source inputs of domestic and industrial origin. Three future scenarios were constructed, by modification of a baseline representative of recent conditions. A climate change scenario, based on simulations by a general circulation model driven by the SRES-A2 scenario of radiative forcing, accounts for an average warming of +3.3 degrees C over the watershed and marked winter increase and summer decrease in precipitation. To illustrate a possible reduction in nitrate pollution from agricultural origin, a scenario of good agricultural practices was considered, introducing catch crops and a 20% decrease in nitrogen fertilisation. Future point source pollution was estimated following the assumptions embedded in scenario SRES-A2 regarding demographic, economic and technologic changes, leading to reductions of 30 to 75% compared to 2000, depending on the pollutants. Four models, addressing separate components of the river system (agronomical model, hydrogeological model, land surface model and water quality model), were used to analyse the relative impact of these scenarios on water quality, in light of their impact on hydrology and crop production. The first-order driving factor of water quality over the 21st century is the projected reduction of point source pollution, inducing a noticeable decrease in eutrophication and oxygen deficits downstream from Paris. The impact of climate change on these terms is driven by the warming of the water column. It enhances algal growth in spring and the loss factors responsible for phytoplankton mortality in late summer (grazers and viruses). In contrast, increased seasonal contrasts in river discharge have a negligible impact on river water quality, as do the changes in riverine nitrate concentration, which never gets limiting. The latter changes have a similar magnitude under the three scenarios. Under climate change, riverine and groundwater nitrate concentrations increase and crop production is advantaged with reduced growing cycles and increased yields. In contrast, nitrate concentrations decrease under the good agricultural practices scenario, with a limited decrease in crop production. When these two scenarios are combined, the changes in nitrate concentrations balance each other and crop yields increase. The results of this numerical exercise indicate that the potential changes to the Seine River system during the 21st century will not lead to severely degraded water quality.

Robustness and precision of an automatic marker detection algorithm for online prostate daily targeting using a standard V-EPID.

An algorithm for the daily localization of the prostate using implanted markers and a standard video-based electronic portal imaging device (V-EPID) has been tested. Prior to planning, three gold markers were implanted in the prostate of seven patients. The clinical images were acquired with a BeamViewPlus 2.1 V-EPID for each field during the normal course radiotherapy treatment and are used off-line to determine the ability of the automatic marker detection algorithm to adequately and consistently detect the markers. Clinical images were obtained with various dose levels from ranging 2.5 to 75 MU. The algorithm is based on marker attenuation characterization in the portal image and spatial distribution. A total of 1182 clinical images were taken. The results show an average efficiency of 93% for the markers detected individually and 85% for the group of markers. This algorithm accomplishes the detection and validation in 0.20-0.40 s. When the center of mass of the group of implanted markers is used, then all displacements can be corrected to within 1.0 mm in 84% of the cases and within 1.5 mm in 97% of cases. The standard video-based EPID tested provides excellent marker detection capability even with low dose levels. The V-EPID can be used successfully with radiopaque markers and the automatic detection algorithm to track and correct the daily setup deviations due to organ motions.

Repression of the defense gene PR-10a by the single-stranded DNA binding protein SEBF.

The potato pathogenesis-related gene PR-10a is transcriptionally activated in response to pathogen infection or elicitor treatment. Characterization of the cis-acting elements of the PR-10a promoter revealed the presence of a silencing element between residues -52 and -27 that contributes to transcriptional regulation. In this study, we have isolated a silencing element binding factor (SEBF) from potato tuber nuclei that binds to the coding strand of the silencing element in a sequence-specific manner. The consensus binding site of SEBF, PyTGTCNC, is present in a number of PR genes and shows striking similarity to the auxin response element. Mutational analysis of the PR-10a promoter revealed an inverse correlation between the in vitro binding of SEBF and the expression of PR-10a. SEBF was purified to homogeneity from potato tubers, and sequencing of the N terminus of the protein led to the isolation of a cDNA clone. Sequence analysis revealed that SEBF is homologous with chloroplast RNA binding proteins that possess consensus sequence-type RNA binding domains characteristic of heterogeneous nuclear ribonucleoproteins (hnRNPs). Overexpression of SEBF in protoplasts repressed the activity of a PR-10a reporter construct in a silencing element-dependent manner, confirming the role of SEBF as a transcriptional repressor.

Direct visualization of protein interactions in plant cells.

The protein NPR1/NIM1 is required for the induction of systemic acquired resistance (SAR) in plants and has been shown to interact with members of the TGA/OBF family of basic leucine zipper (bZIP) transcription factors. However, to date, there is no method available to monitor such interactions in plant cells. We report here an in vivo protein fragment complementation assay (PCA), based on association of reconstituted murine dihydrofolate reductase (mDHFR) with a fluorescent probe to detect protein-protein interaction in planta. We demonstrate that the interaction between Arabidopsis NPR1/NIM1 and the bZIP factor TGA2 is induced by the regulators of SAR, salicylic acid (SA), and its analog 2,6-dichloroisonicotinic acid (INA) with distinct species-specific responses. Furthermore, the induced interaction is localized predominantly in the nucleus. Protein fragment complementation assays could be of value to agricultural research by providing a system for high-throughput biochemical pathway mapping and for screening of small molecules that modulate protein interactions.

PBF-2 is a novel single-stranded DNA binding factor implicated in PR-10a gene activation in potato.

Elicitor-induced activation of the potato pathogenesis-related gene PR-10a requires a 30-bp promoter sequence termed the ERE (elicitor response element) that is bound by the nuclear factor PBF-2 (PR-10a binding factor 2). In this study, PBF-2 has been purified to near homogeneity from elicited tubers through a combination of anion-exchange and DNA affinity chromatography. Evidence demonstrates that inactive PBF-2 is stored in the nuclei of fresh tubers and becomes available for binding to the ERE upon elicitation. A protein with an apparent molecular mass of 24 kD (p24) is a DNA binding component of PBF-2. A cDNA encoding p24 has been cloned and encodes a novel protein with a potential transcriptional activation domain that could also act as a single-stranded DNA binding domain. Both PBF-2 and the cDNA-encoded protein bind with high affinity to the single-stranded form of the ERE in a sequence-specific manner. The inverted repeat sequence of the ERE, TGACAnnnnTGTCA, is critical for binding of this factor in vitro and for PR-10a expression in vivo, supporting the role of PBF-2 as a transcriptional regulator.

Sequential and structural homology between intracellular pathogenesis-related proteins and a group of latex proteins.

The intracellular pathogenesis-related proteins have been identified in a broad range of flowering plants. Some display quite different patterns of expression, in many cases unrelated to the pathogenic response. Nevertheless, these proteins are all very similar and in most cases share more than 35% sequence identity. In this report we investigate the significance of a rather weak similarity between the intracellular pathogenesis-related (IPR or PR-10) proteins and a group of proteins identified in the latex of opium poppy and in Arabidopsis, among others. A sequence analysis held together with the recently published three-dimensional structure of Bet v 1, an IPR protein from birch pollen, strongly suggests sequential and structural homology between the two protein families.

A functional homolog of mammalian protein kinase C participates in the elicitor-induced defense response in potato.

The elicitor-induced activation of the potato pathogenesis-related gene PR-10a is positively controlled by a protein kinase(s) that affects the binding of the nuclear factors PBF-1 (for PR-10a binding factor-1) and PBR-2 to an elicitor response element (ERE). In this study, we have identified a kinase that has properties similar to the conventional isoenzymes of the mammalian protein kinase C (PKC) family. the treatment of potato tuber discs with specific inhibitors of PKC abolished the elicitor-induced binding of the nuclear factor PBF-2 to the ERE. This correlated with a reduction in the accumulation of the PR-10a protein. In contrast, treatment of the tuber discs with 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, led to an increase in binding of PBF-2 to the ERE and the corresponding increase in the level of the PR-10a protein, mimicking the effect seen with the elicitor arachidonic acid. Biochemical characterization of proteins extracted from the particulate fraction of potato tubers demonstrated that a kinase belonging to the conventional isoforms of PKC is present. This was confirmed by immunoprecipitation with antibodies specific to the conventional isoforms of human PKC and in-gel kinase assays. The ability of the immunoprecipitates to phosphorylate the alpha-peptide (a PKC specific substrate) in the presence of the coactivators calcium, phosphatidylserine, and TPA strongly suggested that the immunoprecipitated kinase is similar to the kinase characterized biochemically. Finally, the similar effects of the various modulators of PKC activity on the elicitor-induced resistance against a compatible race of Phytophthora infestans implicate this kinase in the overall defense response in potato.

The starch phosphorylase gene is subjected to different modes of regulation in starch-containing tissues of potato.

Analysis of the levels of starch phosphorylase mRNA and its product in the various organs of the potato plant indicates that the gene is differentially regulated, leading to a high accumulation of the gene product in tubers. The amount of phosphorylase transcripts synthesized in nuclei isolated from tubers and leaves indicates that the difference in the steady-state levels of phosphorylase mRNA in these organs can be explained by different rates of initiation of transcription. However, while rates of initiation of transcription are similar in tubers and stems, the steady-state level of phosphorylase mRNA is much lower in the stem. Transgenic potato plants expressing the beta-glucuronidase (GUS) gene under the control of 5'-flanking sequences of the phosphorylase gene exhibited high levels of GUS activity in petioles, stems, stolons, tubers and roots, but low levels in leaves. This confirms the results of transcription assays observed for leaves, stems and tubers, and indicates that accumulation of phosphorylase mRNA in stems and tubers is not controlled solely by transcription initiation. Finally, histochemical analysis for GUS activity in transgenic potato plants suggests that transcription of the phosphorylase gene predominantly occurs in starch-containing cells associated to vascular tissues, and suggests a role for starch phosphorylase in the mobilization of starch stored along the translocation pathway.

Creation of a Metabolic Sink for Tryptophan Alters the Phenylpropanoid Pathway and the Susceptibility of Potato to Phytophthora infestans.

The creation of artificial metabolic sinks in plants by genetic engineering of key branch points may have serious consequences for the metabolic pathways being modified. The introduction into potato of a gene encoding tryptophan decarboxylase (TDC) isolated from Catharanthus roseus drastically altered the balance of key substrate and product pools involved in the shikimate and phenylpropanoid pathways. Transgenic potato tubers expressing the TDC gene accumulated tryptamine, the immediate decarboxylation product of the TDC reaction. The redirection of tryptophan into tryptamine also resulted in a dramatic decrease in the levels of tryptophan, phenylalanine, and phenylalanine-derived phenolic compounds in transgenic tubers compared with nontransformed controls. In particular, wound-induced accumulation of chlorogenic acid, the major soluble phenolic ester in potato tubers, was found to be two- to threefold lower in transgenic tubers. Thus, the synthesis of polyphenolic compounds, such as lignin, was reduced due to the limited availability of phenolic monomers. Treatment of tuber discs with arachidonic acid, an elicitor of the defense response, led to a dramatic accumulation of soluble and cell wall-bound phenolics in tubers of untransformed potato plants but not in transgenic tubers. The transgenic tubers were also more susceptible to infection after inoculation with zoospores of Phytophthora infestans, which could be attributed to the modified cell wall of these plants. This study provides strong evidence that the synthesis and accumulation of phenolic compounds, including lignin, could be regulated by altering substrate availability through the introduction of a single gene outside the pathway involved in substrate supply. This study also indicates that phenolics, such as chlorogenic acid, play a critical role in defense responses of plants to fungal attack.

Chimeric flavonol sulfotransferases define a domain responsible for substrate and position specificities.

The pFST3 and pFST4' cDNAs encode flavonol sulfotransferases (ST) that are 69% identical in amino acid sequence yet exhibit strict substrate and position specificities. To determine the domain responsible for the properties of the flavonol STs, several chimeric flavonol STs were constructed by the reciprocal exchange of DNA fragments derived from the plasmids pFST3 and pFST4' and by the expression of the corresponding chimeric proteins in Escherichia coli. The chimeric enzymes were enzymatically active even though their activities were reduced compared to the parent enzymes. The specificity of the resulting hybrid proteins indicates that an interval of the flavonol STs spanning amino acids 92-194 of the flavonol 3-ST sequence contains the determinant of the substrate and position preferences. From the comparison of the amino acid sequences between plant and animal STs, this interval can be subdivided into a highly conserved region corresponding to positions 134-152 of the flavonol 3-ST, flanked by two regions of high divergence from 98 to 110 and 153 to 170. In view of the similarities in length and hydropathic profiles as well as the presence of four conserved regions between plant and animal STs, the results of these experiments suggest that this interval is involved in the recognition of substrates and/or catalysis in all STs.

The Activation of the Potato PR-10a Gene Requires the Phosphorylation of the Nuclear Factor PBF-1.

The pathogenesis-related gene PR-10a (formerly STH[middot]2) is induced in various organs of potato after wounding, elicitor treatment, or infection by Phytophthora infestans. Deletion analysis of the promoter of the PR-10a gene enabled us to identify a 50-bp region, located between positions -155 and -105, necessary for the elicitor responsiveness of the [beta]-glucuronidase reporter gene in transgenic potato plants. Within this region, a 30-bp sequence, located between positions -135 and -105, was necessary for the activation of the promoter by the elicitor. However, strong promoter activity after elicitor treatment required the presence of a 20-bp sequence located between positions -155 and -135. The region between -135 and -105 was specifically recognized by two nuclear factors, PBF-1 (PR-10a Binding Factor 1) and PBF-2, and binding of PBF-1 was coordinated with the accumulation of the PR-10a mRNA. Gel shift assays using nuclear extracts pretreated with sodium deoxycholate or alkaline phosphatase suggested that PBF-1 is a multimeric factor in which at least one of the constituent proteins can be phosphorylated. Treatment with alkaline phosphatase also indicated that binding of PBF-1 is positively regulated by phosphorylation and that it is phosphorylated only in tissues in which PR-10a is expressed. The use of protein phosphatase and kinase inhibitors in vivo provided additional evidence that wounding and elicitor treatment induce the phosphorylation of PBF-1 and that this phosphorylation is associated with gene activation.

Redirection of tryptophan leads to production of low indole glucosinolate canola.

Cruciferous plants are known to produce over a hundred different mustard oil glycosides, which are derived from methionine, phenylalanine, or tryptophan. In oil-producing crops like Brassica napus (canola), the presence of indole glucosinolates in seed protein meals has decreased meal palatability and has limited their value as animal feed. We have transformed canola plants with a gene that encodes tryptophan decarboxylase (TDC) in an attempt to redirect tryptophan into tryptamine rather than into indole glucosinolates. Transgenic plants that expressed this decarboxylase activity accumulated tryptamine while correspondingly lower levels of tryptophan-derived indole glucosinolates were produced in all plant parts compared with nontransformed controls. Of particular significance, the indole glucosinolate content of mature seeds from transgenic plants was only 3% of that found in nontransformed seeds. These results demonstrate how the creation of artificial metabolic sinks could divert metabolite flow and be used to remove these undesirable indole glucosinolates, thereby increasing the value of the oilseed meals, which are produced after extraction of oil from the seed.

Transgenic potato plants overexpressing the pathogenesis-related STH-2 gene show unaltered susceptibility to Phytophthora infestans and potato virus X.

The STH-2 gene is rapidly activated in potato leaves and tubers following elicitation or infection by Phytophthora infestans. However, its biochemical function remains unknown. In order to ascertain if STH-2 protein is directly involved in the defense of potato against pathogens, the STH-2 coding sequence under the control of the CaMV 35S promoter was introduced into potato plants. Transgenic plants expressing the STH-2 gene were analyzed for an altered pattern of susceptibility to a compatible race of P. infestans and to potato virus X. Results indicate that constitutive expression of the STH-2 gene did not reduce susceptibility of potato to these pathogens.

Identification of cis-acting elements involved in the regulation of the pathogenesis-related gene STH-2 in potato.

We have characterized a genomic clone containing the potato pathogenesis-related genes STH-2 and STH-21. The two genes are found 4 kb apart on the same chromosome and their sequences are highly similar. They present the same transcriptional orientation and are both interrupted by a single intron. A chimaeric gene consisting of 1015 bp of 5'-flanking sequence and part of the first exon of STH-2 fused to the bacterial beta-glucuronidase gene was highly-expressed in tubers of transgenic potato plants after wounding and elicitor treatments. The levels of activity observed in these transgenic plants parallel those observed for the accumulation of STH-2 mRNAs under similar conditions. This indicates that cis-acting elements necessary for the proper activation of the gene are present within 1 kb of 5'-flanking sequences. Functional analysis of 5' deletions of the STH-2/GUS constructs by transient expression in leaf protoplasts revealed the presence of an upstream regulatory sequence between -135 and -52 which contains a TGAC motif, and a possible negative regulatory region between -52 and -28. A factor present in nuclear extracts of wounded potato tubers was found to bind specifically to nucleotides located between -135 to -105, suggesting that this region contains important cis-regulatory elements.

Molecular characterization of two plant flavonol sulfotransferases.

cDNA clones coding for flavonol 3- and 4'-sulfotransferases (STs) were isolated by antibody screening of a cDNA expression library produced from poly(A)+ RNA extracted from terminal buds of Flaveria chloraefolia. Sequence analysis revealed full-length cDNA clones with open reading frames of 933 and 960 base pairs, which encode polypeptides containing 311 and 320 amino acids, respectively. This corresponds to a molecular mass of 36,442 Da for the 3-ST and 37,212 Da for the 4'-ST. Expression of these clones in Escherichia coli led to the synthesis of beta-galactosidase-ST fusion proteins having the same substrate and position specificities as those for the 3- and 4'-flavonol ST enzymes isolated from the plant. Comparison of the deduced amino acid sequence of the two clones revealed an overall identity of 69% in 311 amino acid residues. The two flavonol STs of F. chloraefolia also shared significant sequence similarities with steroid and aryl STs found in animal tissues and with the senescence marker protein 2 isolated from rat liver, suggesting an evolutionary link between plant and animal STs.

The defense-related STH-2 gene product of potato shows race-specific accumulation after inoculation with low concentrations of Phytophthora infestans zoospores.

The defense-related STH-2 gene is rapidly activated following infection or elicitor treatment of potato (Solanum tuberosum L.) tubers. However, its physiological or biochemical function is unknown. To study the STH-2 gene product and its accumulation during the defense response, we raised antibodies to a ß-galactosidase-STH-2 fusion protein in Escherichia coli. The antiserum specifically recognized a protein of the predicted 17-kDa size in extracts of elicited tuber disks when analyzed by Western blot. In control extracts this band was not detected. The accumulation of STH-2 protein in response to incompatible and compatible zoospores of Phytophthora infestans (Mont.) de Bary depended on the inoculum density applied. Whereas a low concentration of spores induced accumulation of STH-2 protein faster in the incompatible than the compatible interaction, this difference in timing was less pronounced at higher inoculum densities. Inoculation with a high concentration of compatible spores also resulted in the disappearance of STH-2 protein late during the infection. In both control and induced tuber tissue the antibody strongly reacted with an unknown protein of 18 kDa. This protein was present constitutively in tubers, but in leaves its accumulation was stimulated by inoculation with P. infestans.

High levels of tryptamine accumulation in transgenic tobacco expressing tryptophan decarboxylase.

A full-length complementary DNA clone encoding tryptophan decarboxylase (TDC; EC 4.1.1.28) from Catharanthus roseus (De Luca V, Marineau C, Brisson N [1989] Proc Natl Acad Sci USA 86: 2582-2586) driven by the CaMV 35S promoter was introduced into tobacco (Nicotiana tabacum) to direct the synthesis of the protoalkaloid tryptamine from endogenous tryptophan. Young, fully expanded leaves of CaMV 35S-TDC transformed plants had from four to 45 times greater TDC activity than did controls. Tryptamine accumulated in transgenic plants to levels that were directly proportional to their TDC specific activity. Despite their increased tryptamine content, the growth and development of the CaMV 35S-TDC plants appeared normal with no significant differences in indole-3-acetic acid levels between high tryptamine and control plants. Plants with the highest TDC activity contained more than 1 milligram of tryptamine per gram fresh weight, a 260-fold increase over controls.