Kent F. McCue

Effect of temperature on expression of genes encoding enzymes for starch biosynthesis in developing wheat endosperm

The effect of high temperature on starch accumulation, starch granule populations, and expression of genes encoding key enzymes for starch biosynthesis was examined during grain development in wheat (Triticum aestivum L. cv. Butte 86). High temperature applied from anthesis to maturity reduced the duration of starch accumulation. Starch accumulation ceased approximately 6 days earlier for grain produced under a 37/17 8C (day/night) regimen and 21 days earlier under a 37/28 8C (day/night) regimen than for grain produced under a 24/17 8C (day/night) regimen. Compared to the 24/17 8C regimen, starch content was approximately 19% less for mature grain produced under the 37/17 8C regimen and 58% less under the 37/28 8C regimen. Based on relative volume, the smaller type B starch granules were the predominant class in mature grain produced under the 24/17 and 37/17 8C regimens, whereas the larger type A granules were predominant in grain produced under the 37/28 8C regimen. Under the 24/17 8C regimen, steady state transcript levels for ADP-glucose pyrophosphorylase, starch synthases I, II, and III, granule-bound starch synthase, and starch branching enzymes I and II were highest from 12/16 days post-anthesis (dpa). Under the 37/17 8C regimen, steady state levels of these transcripts followed the same temporal pattern, but were substantially lower. Under the 37/28 8C regimen, transcript levels peaked earlier, at 7 dpa. The high temperature regimens reduced the relativ el evels of transcripts for starch synthase more than the other starch biosynthetic enzymes. Published by Elsevier Science Ireland Ltd.

Metabolic engineering of glycine betaine synthesis : plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance

Certain higher plants synthesize and accumulate glycine betaine, a compound with osmoprotectant properties. Biosynthesis of glycine betaine proceeds via the pathway choline → betaine aldehyde → glycine betaine. Plants such as tobacco (Nicotiana tabacum L.) which do not accumulate glycine betaine lack the enzymes catalyzing both reactions. As a step towards engineering glycine betaine accumulation into a non-accumulator, spinach and sugar beet complementary-DNA sequences encoding the second enzyme of glycine-betaine synthesis (betaine aldehyde dehydrogenase, BADH, EC 1.2.1.8) were expressed in tobacco. Despite the absence of a typical transit peptide, BADH was targeted to the chloroplast in leaves of transgenic plants. Levels of extractable BADH were comparable to those in spinach and sugar beet, and the molecular weight, isoenzyme profile and Km for betaine aldehyde of the BADH enzymes from transgenic plants were the same as for native spinach or sugar beet BADH. Transgenic plants converted supplied betaine aldehyde to glycine betaine at high rates, demonstrating that they were able to transport betaine aldehyde across both the plasma membrane and the chloroplast envelope. The glycine betaine produced in this way was not further metabolized and reached concentrations similar to those in plants which accumulate glycine betaine naturally. Betaine aldehyde was toxic to non-transformed tobacco tissues whereas transgenic tissues were resistant due to detoxification of betaine aldehyde to glycine betaine. Betaine aldehyded ehydrogenase is therefore of interest as a potential selectable marker, as well as in the metabolic engineering of osmoprotectant biosynthesis.

Gene Rpi-bt1 from Solanum bulbocastanum Confers Resistance to Late Blight in Transgenic Potatoes

Phytophthora infestans, the causal agent of late blight is the most devastating pathogen of cultivated potato worldwide. Utilizing map based cloning; a genomic region containing a cluster of six nucleotide binding site-leucine-rich repeat resistance gene analogs was isolated from the wild potato species Solanum bulbocastanum. Four genes were pseudogenes, with coding sequences interrupted by either frame shift mutations or premature stop codons. However, neither of the two uninterrupted genes conferred resistance to P. infestans when introduced into susceptible potatoes. Specific primers for one of the pseudogenes were used to amplify an uninterrupted cDNA from P. infestans-infected S. bulbocastanum leaves. A corresponding gDNA was amplified from a late blight-resistant bulbocastanum–tuberosum introgression line (Rpi-bt1). The Rpi-bt1 gene under transcriptional control of the constitutive potato Ubi3 promoter was found to confer resistance to P. infestans in several transgenic potato lines in a whole plant greenhouse assay.ResumenPhytophthora infestans, el agente causal del tizón tardío, es el patógeno mas devastador de la papa cultivada en el mundo. Mediante la clonación basada en mapas, se aisló de la especie silvestre de papa Solanum bulbocastanum una región genómica que incluía un grupo de repetición con seis análogos de genes de resistencia de sitios de unión de nucleótidos ricos en leucina. Cuatro genes fueron pseudogenes, con secuencias de codificación interrumpidas ya fuera por mutaciones por cambio de marcos o por codones de terminación prematura. No obstante, ninguno de los dos genes sin interrupción confirió resistencia a P. infestans cuando se introdujeron a papas susceptibles. Se utilizaron iniciadores específicos para uno de los pseudogenes para amplificar un ADNc ininterrumpido de hojas de S. bulbocastanum infectadas con P. infestans. También se amplificó un ADNg correspondiente de una línea de introgresión (Rpi-bt1) de bulbocastanum-tuberosum resistente al tizón tardío. Se encontró que el gen Rpi-bt1 bajo control transcripcional del promotor de papa Ubi3 confiere resistencia a P. infestans en varias líneas de papa transgénica en un ensayo con planta completa bajo invernadero.

pBINPLUS/ARS: an improved plant transformation vector based on pBINPLUS

Binary plant transformation vectors are widely used for introduction of transgenes into plants via Agrobacterium tumefaciens-mediated transformation. We report the construction of a binary plant vector pBINPLUS/ARS based on the pBINPLUS vector. Improvements introduced into pBINPLUS/ARS include the use of nonproprietary (ubiquitin-3 gene of Solanum tuberosum) promoter and terminator sequences for transcription of the NptII selectable marker and introduction of rare 8-bp restriction enzyme sites flanking both the NptII coding sequence (PmeI) and the entire selectable marker gene (FseI). This vector offers all of the advantages of its predecessor pBINPLUS and its helper plasmid pUCAP, which use the proprietary nopaline synthase promoter and terminator, while allowing for facile modification of selectable marker sequences in complex binary vector constructs. pBINPLUS/ARS has been used to introduce transgenes into potato and other crop species and is available to all researchers in academic, government, and industrial laboratories for proof-of-principle and commercial applications.

Starch-branching enzymesSbe1 andSbe2 from wheat (Triticum aestivum cv. Cheyenne): Molecular characterization, development expression, and homoeologue assignment by differential PCR

Starch is the main component of the wheat kernel, and wheat flour is used for hundreds of food and nonfood products. We are exploring ways to improve wheat quality and to develop new uses for wheat based on altered starch characteristics. To understand the molecular basis for variations in the physical and chemical properties of starch, we examined transcripts for starch biosynthetic enzymes. cDNAs encoding 2 isoforms of starch-branching enzyme (Sbe1, Sbe2) were isolated from wheat endosperm. The longestSbe1 andSbe2 cDNAs were 2797 and 2975 bp, respectively, and they shared extensive identity withSbe sequences reported for wheat and other species. With orthologue-specific primer pairs, homoeologue assignments to chromosome 7 were made forSbe1#19 (TRIae: Sbe1A.1) andSbe1#9 (TRIae:Sbe1D.1) using the wheat cv. Chinese Spring nullisomic-tetrasomic-7 lines. This strategy may prove useful for future mapping of expressed sequence tag (EST) data. TheSbe cDNAs and a granule-bound starch synthase cDNA (GbssI) (from an EST sequencing project) were used to examine the steady-state RNA levels during development of the wheat. Steady-state levels ofSbe2 mRNA were detectable 5 d postanthesis (DPA) and reached a maximum at 10 DPA. Steady-state levels ofSbe1 andGbssI began to rise at 10 DPA and peaked at 15 DPA. Levels of all messages declined rapidly at 20–25 DPA. Reported here is the first analysis of transcripts for these enzymes in the same RNA pools and demonstration that expression patterns are unique and developmentally regulated.

Structure of Two Solanum bulbocastanum Polyubiquitin Genes and Expression of Their Promoters in Transgenic Potatoes

Two polyubiquitin genes, bul409 and bul427, were isolated from a Solanum bulbocastanum BAC library. The bul409 and bul427 genes encode hexameric and heptameric polyproteins, respectively. bul427 exhibits a number of features suggestive of a pseudogene: (1) The last ubiquitin monomer of bul427 is interrupted by a frame shift mutation. (2) The coding sequence is flanked 3′ by mitochondrial and chloroplast sequences and 5′ by a protein kinase pseudogene. However, characterization of cDNAs amplified using bul427-based primers demonstrated that bul427 is transcriptionally active. Chimeric transgenes encoding β-glucuronidase (GUS) translationally fused to the first ubiquitin-coding units of bul409, a truncated version 409s, and bul427 were constructed and introduced into potato. In transgenic potato lines both S. bulbocastanum promoters were weakly transcribed in tubers and efficiently transcribed in leaves. In leaves, bul409s was wound-induced, while bul427 was not. In tubers both promoters were wound-induced. In unwounded leaves and tubers, the steady state mRNA levels from both bul promoters were lower than the steady state mRNA levels from the Cauliflower Mosaic Virus 35S promoter. However, in the leaves and tubers of many of the transgenic lines the GUS activity was significantly higher in the bul lines compared to the 35S lines. The apparent inconsistency of higher enzymatic activity correlated with lower steady state levels of mRNA demonstrates the enhanced protein expression observed with ubiquitin fusion proteins.ResumenDos genes de poliubiquitina, bul409 y bul427, fueron aislados de una librería BAC de Solanum bulbocastanum. Los genes bul409 y bul427 codifican poliproteínas hexámera y heptámera, respectivamente. El bul427 exhibe varios rasgos que sugieren un seudo gen: (1) El último monómero de bul427 es interrumpido por una mutación con desplazamiento de la pauta de lectura. (2) La secuencia codificante es flanqueada en 3′ por secuencias mitocondriales y de cloroplastos y en 5′ por un seudogen de proteína quinasa. Sin embargo, la caracterización de cDNAs amplificadas usando iniciadores basados en bul427 demostró que bul427 es transcripcionalmente activo. Se construyeron e introdujeron en papa transgenes quiméricos que codifican β-glucuronidasa (GUS) translacionalmente fusionados a las primeras unidades que codifican ubiquitina de bul409, una versión truncada de 409s y bul427. En las líneas de papa transgénica de S. bulbocastanum, los promotores fueron débilmente transcritos en los tubérculos y eficientemente transcritos en hojas. En hojas el bul409s fue inducido por heridas, mientras que bul427 no fue. En tubérculos, ambos promotores fueron inducidos por heridas. En hojas y tubérculos sin herir, los niveles mRNA en estado de equilibrio para ambos promotores bul fueron más bajos que los niveles de mRNA en estado de equilibrio del promotor 35S del virus del Mosaico de la Coliflor. Sin embargo, en hojas y tubérculos de muchas líneas transgénicas, la actividad de GUS fue significativamente más alta en las líneas bul en comparación con las 35S. La aparente inconsistencia de una actividad enzimática mayor, correlacionada con bajos niveles de mRNA en estado de equilibrio demuestra el incremento de la expresión de proteína observada con proteínas fusión ubiquitina.

Modifying glycoalkaloid content in transgenic potato - Metabolome impacts

Metabolite profiling has been used to assess the potential for unintended composition changes in potato (Solanum tuberosum L. cv. Desirée) tubers, which have been genetically modified (GM) to reduce glycoalkaloid content, via the independent down-regulation of three genes SGT1, SGT2 and SGT3 known to be involved in glycoalkaloid biosynthesis. Differences between the three groups of antisense lines and control lines were assessed using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC)-MS, and data analysed using principal component analysis and analysis of variance. Compared with the wild-type (WT) control, LC-MS revealed not only the expected changes in specific glycoalkaloid levels in the GM lines, but also significant changes in several other metabolites, some of which were explicable in terms of known pathways. Analysis of polar and non-polar metabolites by GC-MS revealed other significant (unintended) differences between SGT lines and the WT, but also between the WT control and other control lines used.

Compositional and toxicological analysis of a GM potato line with reduced α-solanine content--a 90-day feeding study in the Syrian Golden hamster.

Steroidal glycoalkaloids (GAs) are toxins, produced by plants of the Solanaceae family. The potato plant (Solanum tuberosum L.) and its tubers predominantly contain the two GAs α-chaconine and α-solanine. These compounds are believed to act in synergy, and the degree of toxicity may therefore depend on their ratio in the potato. To determine the influence of α-solanine: α-chaconine ratio in potatoes on toxicity, a GM potato line (SGT 9-2) with reduced α-solanine content, and the parental control line (Desirée wild-type) having a traditional α-solanine: α-chaconine ratio were (1) studied for compositional similarity by analysing for a range of potato constituents, and (2) used in a 90-day feeding trial with the Syrian Golden hamster to study differential toxicity. The animal feeding study used diets with up to 60% freeze-dried potato powder from either line. Whilst data indicated some compositional differences between the GM line and its wildtype control these did not raise concerns related to nutritional value or safety. Results of the feeding trials showed a low number of significant differences between potato lines with different α-solanine: α-chaconine ratio but none were considered to raise safety concerns with regard to human (or animal) consumption.

Impact of light-exposure on the metabolite balance of transgenic potato tubers with modified glycoalkaloid biosynthesis.

Metabolite profiling (liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC-MS)) was used to assess the impact of light on the composition of transgenic potato (Solanum tuberosum L. cv. Desirée) with reduced glycoalkaloid content via the down-regulation of the SGT1 gene. Transgenic tubers exhibited an almost complete knock-out of α-solanine production and light had little impact on its accumulation. Levels of α-chaconine increased significantly in the peel of both the control and transgenic lines when exposed to light, particularly in the transgenic line. Major differences in metabolite profiles existed between outer and inner tuber tissues, and between light and dark-treated tubers. Many of the light-induced changes are explicable in terms of pathways known to be affected by stress responses. The impact of transgenesis on profiles was much less than that of tissue type or light and most differences were explicable in terms of the modification to the glycoalkaloid pathway.

Accurate measurement of transgene copy number in crop plants using droplet digital PCR.

Genetic transformation is a powerful means for the improvement of crop plants, but requires labor- and resource-intensive methods. An efficient method for identifying single-copy transgene insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time-consuming and often requires a large amount of genomic DNA and radioactively labeled probes. Alternatively, qPCR requires less DNA and is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one- and two-copy events in transgenic plants with large genomes. To address this need, we developed a droplet digital PCR-based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato and wheat. The method utilizes specific primers to amplify target transgenes, and endogenous reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence-specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species.