Ellen N. Friel

Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

BackgroundWe describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes.ResultsWe give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs.ConclusionIn developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

A Genomics Approach Reveals That Aroma Production in Apple Is Controlled by Ethylene Predominantly at the Final Step in Each Biosynthetic Pathway

Ethylene is the major effector of ripening in many fleshy fruits. In apples (Malus x domestica) the addition of ethylene causes a climacteric burst of respiration, an increase in aroma, and softening of the flesh. We have generated a transgenic line of ‘Royal Gala’ apple that produces no detectable levels of ethylene using antisense ACC OXIDASE, resulting in apples with no ethylene-induced ripening attributes. In response to external ethylene these antisense fruits undergo a normal climacteric burst and produced increasing concentrations of ester, polypropanoid, and terpene volatile compounds over an 8-d period. A total of 186 candidate genes that might be involved in the production of these compounds were mined from expressed sequence tags databases and full sequence obtained. Expression patterns of 179 of these were assessed using a 15,720 oligonucleotide apple microarray. Based on sequence similarity and gene expression patterns we identified 17 candidate genes that are likely to be ethylene control points for aroma production in apple. While many of the biosynthetic steps in these pathways were represented by gene families containing two or more genes, expression patterns revealed that only a single member is typically regulated by ethylene. Only certain points within the aroma biosynthesis pathways were regulated by ethylene. Often the first step, and in all pathways the last steps, contained enzymes that were ethylene regulated. This analysis suggests that the initial and final enzymatic steps with the biosynthetic pathways are important transcriptional regulation points for aroma production in apple.

An alcohol acyl transferase from apple (cv. Royal Gala), MpAAT1, produces esters involved in apple fruit flavor

Apple flavor is characterized by combinations of ester compounds, which increase markedly during fruit ripening. The final step in ester biosynthesis is catalyzed by alcohol acyl transferases (AATs) that use coenzyme A (CoA) donors together with alcohol acceptors as substrates. The gene MpAAT1, which produces a predicted protein containing features of other plant acyl transferases, was isolated from Malus pumila (cv. Royal Gala). The MpAAT1 gene is expressed in leaves, flowers and fruit of apple. The recombinant enzyme can utilize a range of alcohol substrates from short to medium straight chain (C3–C10), branched chain, aromatic and terpene alcohols. The enzyme can also utilize a range of short to medium chain CoAs. The binding of the alcohol substrate is rate limiting compared with the binding of the CoA substrate. Among different alcohol substrates there is more variation in turnover compared with Km values. MpAAT1 is capable of producing many esters found in Royal Gala fruit, including hexyl esters, butyl acetate and 2‐methylbutyl acetate. Of these, MpAAT1 prefers to produce the hexyl esters of C3, C6 and C8 CoAs. For the acetate esters, however, MpAAT1 preference depends upon substrate concentration. At low concentrations of alcohol substrate the enzyme prefers utilizing the 2‐methylbutanol over hexanol and butanol, while at high concentrations of substrate hexanol can be used at a greater rate than 2‐methylbutanol and butanol. Such kinetic characteristics of AATs may therefore be another important factor in understanding how the distinct flavor profiles of different fruit are produced during ripening.

2 – Avocado Oil

Publisher Summary Avocado (Persea americana Mil.) is a subtropical tree that is relatively frost-sensitive and grows to a height of 5–30 m. The fleshy fruit are borne yearly from the current seasons wood and the green fruit ripen only after being harvested. Avocado oil is obtained from the flesh fruit and is a relatively new arrival in culinary circles. The predominant uses of avocado oil are in the cosmetic industry because of its stability and high level of vitamin E (α-tocopherol). The bulk of avocado oil is extracted by relatively harsh methods like high temperature and solvent extraction, typically followed by standard refining steps. The ultimate market of the oil generally dictates the fruit quality required. For the production of good-quality cold-pressed oil, the fruit must be relatively sound with mainly cosmetic-quality issues or too small for sale. Cold-pressed avocado oil is a new product with significant production, commercialization, and marketing only occurring in the 21st century. Avocado oil is a high-value oil with excellent qualities for both culinary and cosmetic uses.

Pathway analysis in horticultural crops: linalool as an example

Abstract Linalool is an important chiral compound in the fragrance industry and is present in many products. Although, linalool has also been found in the fruit of kiwifruit and apple it is more abundant in the flowers, where it plays a key role as an intermediate to a number of interesting fragrance compounds. Three genes found to catalyse the production of linalool from geranyl diphosphate, have been mined from the HortResearch Plant EST database. The function of these genes has been proven using heterologous over-expression technologies. The similarities and differences between our genes and those already published are highlighted. Finally, we show the diversity in the fate of linalool in species of kiwifruit and apple, with discussion of the genes involved.