Ian M. Prosser

Aphid alarm pheromone produced by transgenic plants affects aphid and parasitoid behavior

The alarm pheromone for many species of aphids, which causes dispersion in response to attack by predators or parasitoids, consists of the sesquiterpene (E)-β-farnesene (Eβf). We used high levels of expression in Arabidopsis thaliana plants of an Eβf synthase gene cloned from Mentha × piperita to cause emission of pure Eβf. These plants elicited potent effects on behavior of the aphid Myzus persicae (alarm and repellent responses) and its parasitoid Diaeretiella rapae (an arrestant response). Here, we report the transformation of a plant to produce an insect pheromone and demonstrate that the resulting emission affects behavioral responses at two trophic levels.

Isolation of a cDNA from Saccharomyces cerevisiae that encodes a high affinity sulphate transporter at the plasma membrane.

Resistance to selenate and chromate, toxic analogues of sulphate, was used to isolate a mutant of Saccharomyces cerevisiae deficient in the capacity to transport sulphate into the cells. A clone which complements this mutation was isolated from a cDNA library prepared from S. cerevisiae poly(A)+ RNA. This clone contains an insert which is 2775 by in length and has a single open reading frame that encodes a 859 amino acid polypeptide with a molecular mass of 96 kDa. Sequence motifs within the deduced amino acid sequence of this cDNA (SUL1) show homology with conserved areas of sulphate transport proteins from other organisms. Sequence analysis predicts the position of 12 putative membrane spanning domains in SUL1. When the cDNA for SUL1 was expressed in S. cerevisiae, a high affinity sulphate uptake activity (Km = 7.5 ± 0.6 μM for SO42−) was observed. A genomic mutant of S. cerevisiae in which 1096 by were deleted from the SUL1 coding region was constructed. This mutant was unable to grow on media containing less than 5 mM sulphate unless complemented with a plasmid containing the SUL1 cDNA. We conclude that the SUL1 cDNA encodes a S. cerevisiae high affinity sulphate transporter that is responsible for the transfer of sulphate across the plasma membrane from the external medium.

Molecular cloning and characterisation of asparagine synthetase from Lotus japonicus: Dynamics of asparagine synthesis in N-sufficient conditions

Two cDNA clones, LJAS1 and LJAS2, encoding different asparagine synthetases (AS) have been identified and sequenced and their expression in Lotus japonicus characterised. Analysis of predicted amino acid sequences indicated a high level of identity with other plant AS sequences. No other AS genes were detected in the L. japonicus genome. LJAS1 gene expression was found to be root-enhanced and lower levels of transcript were also identified in photosynthetic tissues. In contrast, LJAS2 gene expression was root-specific. These patterns of AS gene expression are different from those seen in pea. AS gene expression was monitored throughout a 16 h light/8 h dark day, under nitrate-sufficient conditions. Neither transcript showed the dark-enhanced accumulation patterns previously reported for other plant AS genes. To evaluate AS activity, the molecular dynamics of asparagine synthesis were examined in vivo using 15N-ammonium labelling. A constant rate of asparagine synthesis in the roots was observed. Asparagine was the most predominant amino-component of the xylem sap and became labelled at a slightly slower rate than the asparagine in the roots, indicating that most root asparagine was located in a cytoplasmic ‘transport’ pool rather than in a vacuolar ‘storage’ pool. The steady-state mRNA levels and the 15N-labelling data suggest that light regulation of AS gene expression is not a factor controlling N-assimilation in L. japonicus roots during stable growth in N-sufficient conditions.

Expression profiling of potato germplasm differentiated in quality traits leads to the identification of candidate flavour and texture genes

Quality traits such as flavour and texture are assuming a greater importance in crop breeding programmes. This study takes advantage of potato germplasm differentiated in tuber flavour and texture traits. A recently developed 44 000-element potato microarray was used to identify tuber gene expression profiles that correspond to differences in tuber flavour and texture as well as carotenoid content and dormancy characteristics. Gene expression was compared in two Solanum tuberosum group Phureja cultivars and two S. tuberosum group Tuberosum cultivars; 309 genes were significantly and consistently up-regulated in Phureja, whereas 555 genes were down-regulated. Approximately 46% of the genes in these lists can be identified from their annotation and amongst these are candidates that may underpin the Phureja/Tuberosum trait differences. For example, a clear difference in the cooked tuber volatile profile is the higher level of the sesquiterpene α-copaene in Phureja compared with Tuberosum. A sesquiterpene synthase gene was identified as being more highly expressed in Phureja tubers and its corresponding full-length cDNA was demonstrated to encode α-copaene synthase. Other potential ‘flavour genes’, identified from their differential expression profiles, include those encoding branched-chain amino acid aminotransferase and a ribonuclease suggesting a mechanism for 5′-ribonucleotide formation in potato tubers on cooking. Major differences in the expression levels of genes involved in cell wall biosynthesis (and potentially texture) were also identified, including genes encoding pectin acetylesterase, xyloglucan endotransglycosylase and pectin methylesterase. Other gene expression differences that may impact tuber carotenoid content and tuber life-cycle phenotypes are discussed.

NADP-linked malic enzyme and malate metabolism in ageing tomato fruit

Abstract Malate dehydrogenase (oxaloacetate-decarboxylating) (NADP + ) EC 1.1.1.40, malic enzyme, has been purified 40-fold to a homogeneous state using affinity chromatography and gel permeation chromatography. The M r is 260–265 K with four subunits each of 64–65 K. The enzyme has some competitive or non-competitive inhibitors, particularly some of the Krebs cycle acids and exhibits a rapid rise in activity at the same time as activity of the enzymes of the Krebs cycle are decreasing in the tomato mitochrondrion. The malic enzyme is restricted to the cytosol. The relevance of this information to malate metabolism in plants is discussed.

(+)-(10R)-Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis.

Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of (3)H- and (2)H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.

Heterologous expression and transcript analysis of gibberellin biosynthetic genes of grasses reveals novel functionality in the GA3ox family

BackgroundThe gibberellin (GA) pathway plays a central role in the regulation of plant development, with the 2-oxoglutarate-dependent dioxygenases (2-ODDs: GA20ox, GA3ox, GA2ox) that catalyse the later steps in the biosynthetic pathway of particularly importance in regulating bioactive GA levels. Although GA has important impacts on crop yield and quality, our understanding of the regulation of GA biosynthesis during wheat and barley development remains limited. In this study we identified or assembled genes encoding the GA 2-ODDs of wheat, barley and Brachypodium distachyon and characterised the wheat genes by heterologous expression and transcript analysis.ResultsThe wheat, barley and Brachypodium genomes each contain orthologous copies of the GA20ox, GA3ox and GA2ox genes identified in rice, with the exception of OsGA3ox1 and OsGA2ox5 which are absent in these species. Some additional paralogs of 2-ODD genes were identified: notably, a novel gene in the wheat B genome related to GA3ox2 was shown to encode a GA 1-oxidase, named as TaGA1ox-B1. This enzyme is likely to be responsible for the abundant 1β-hydroxylated GAs present in developing wheat grains. We also identified a related gene in barley, located in a syntenic position to TaGA1ox-B1, that encodes a GA 3,18-dihydroxylase which similarly accounts for the accumulation of unusual GAs in barley grains. Transcript analysis showed that some paralogs of the different classes of 2-ODD were expressed mainly in a single tissue or at specific developmental stages. In particular, TaGA20ox3, TaGA1ox1, TaGA3ox3 and TaGA2ox7 were predominantly expressed in developing grain. More detailed analysis of grain-specific gene expression showed that while the transcripts of biosynthetic genes were most abundant in the endosperm, genes encoding inactivation and signalling components were more highly expressed in the seed coat and pericarp.ConclusionsThe comprehensive expression and functional characterisation of the multigene families encoding the 2-ODD enzymes of the GA pathway in wheat and barley will provide the basis for a better understanding of GA-regulated development in these species. This analysis revealed the existence of a novel, endosperm-specific GA 1-oxidase in wheat and a related GA 3,18-dihydroxylase enzyme in barley that may play important roles during grain expansion and development.

Cloning and characterisation of a prenyltransferase from the aphid Myzus persicae with potential involvement in alarm pheromone biosynthesis

The majority of aphid species release an alarm pheromone with the most common component being the sesquiterpene (E)‐β‐farnesene, sometimes accompanied by other sesquiterpenes or monoterpenes. The genes/enzymes involved in the production of these compounds have not been identified in aphids although some components of isoprenoid biosynthesis have been identified in other insect species. Here we report the cloning, expression and characterisation of a prenyltransferase from the aphid Myzus persicae which can act as a farnesyl pyrophosphate synthase or a geranyl pyrophosphate synthase to produce both sesquiterpenes and monoterpenes and hence could be responsible for the biosynthesis of the observed components of the alarm pheromones. In addition, the enzyme can utilise geranyl pyrophosphate to produce farnesyl pyrophosphate showing that the synthesis of the latter involves the sequential condensation of isoprenyl pyrophosphate units.

Overexpression of GCN2‐type protein kinase in wheat has profound effects on free amino acid concentration and gene expression

A key point of regulation of protein synthesis and amino acid homoeostasis in eukaryotes is the phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) by protein kinase general control nonderepressible (GCN)-2. In this study, a GCN2-type PCR product (TaGCN2) was amplified from wheat (Triticum aestivum) RNA, while a wheat eIF2α homologue was identified in wheat genome data and found to contain a conserved target site for phosphorylation by GCN2. TaGCN2 overexpression in transgenic wheat resulted in significant decreases in total free amino acid concentration in the grain, with free asparagine concentration in particular being much lower than in controls. There were significant increases in the expression of eIF2α and protein phosphatase PP2A, as well as a nitrate reductase gene and genes encoding phosphoserine phosphatase and dihydrodipicolinate synthase, while the expression of an asparagine synthetase (AS1) gene and genes encoding cystathionine gamma-synthase and sulphur-deficiency-induced-1 all decreased significantly. Sulphur deficiency-induced activation of these genes occurred in wild-type plants but not in TaGCN2 overexpressing lines. Under sulphur deprivation, the expression of genes encoding aspartate kinase/homoserine dehydrogenase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase was also lower than in controls. The study demonstrates that TaGCN2 plays an important role in the regulation of genes encoding enzymes of amino acid biosynthesis in wheat and is the first to implicate GCN2-type protein kinases so clearly in sulphur signalling in any organism. It shows that manipulation of TaGCN2 gene expression could be used to reduce free asparagine accumulation in wheat grain and the risk of acrylamide formation in wheat products.

Approaches to cloning genes encoding for nutrient transporters in plants

A number of strategies for identifying and cloning genes involved in nutrient transport and assimilation processes are discussed. Particular attention is paid to phenotypic screening by heterologous complementation of mutations which affect transport processes in other organisms and use of insertion elements in plant genomes.