Anthocyanin concentration is an important determinant of the colour of many fruits. In apple (Malus x domestica), centuries of breeding have produced numerous varieties in which levels of anthocyanin pigment vary widely and change in response to environmental and developmental stimuli. The apple fruit cortex is usually colourless, although germplasm does exist where the cortex is highly pigmented due to the accumulation of either anthocyanins or carotenoids. From studies in a diverse array of plant species, it is apparent that anthocyanin biosynthesis is controlled at the level of transcription. Here we report the transcript levels of the anthocyanin biosynthetic genes in a red-fleshed apple compared with a white-fleshed cultivar. We also describe an apple MYB transcription factor, MdMYB10, that is similar in sequence to known anthocyanin regulators in other species. We further show that this transcription factor can induce anthocyanin accumulation in both heterologous and homologous systems, generating pigmented patches in transient assays in tobacco leaves and highly pigmented apple plants following stable transformation with constitutively expressed MdMYB10. Efficient induction of anthocyanin biosynthesis in transient assays by MdMYB10 was dependent on the co-expression of two distinct bHLH proteins from apple, MdbHLH3 and MdbHLH33. The strong correlation between the expression of MdMYB10 and apple anthocyanin levels during fruit development suggests that this transcription factor is responsible for controlling anthocyanin biosynthesis in apple fruit; in the red-fleshed cultivar and in the skin of other varieties, there is an induction of MdMYB10 expression concurrent with colour formation during development. Characterization of MdMYB10 has implications for the development of new varieties through classical breeding or a biotechnological approach.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2006.02964.x

Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10

http://www.medicinalgenomics.com/wp-content/uploads/2011/12/Monoterpene-and-sesquiterpene-synthases-and-the-origin-of-terpene-skeletal.pdf

Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants.

Agro-infiltration of leaf tissue with binary vectors harbouring a sequence of interest is a rapid method of expressing proteins in plants. It has recently been shown that flanking the sequence to be expressed with a modified 5'-untranslated region (UTR) and the 3'-UTR from Cowpea mosaic virus (CPMV) RNA-2 (CPMV-HT) within the binary vector pBINPLUS greatly enhances the level of expression that can be achieved [Sainsbury, F. and Lomonossoff, G.P. (2008)Plant Physiol. 148, 1212-1218]. To exploit this finding, a series of small binary vectors tailored for transient expression (termed the pEAQ vectors) has been created. In these, more than 7 kb of non-essential sequence was removed from the pBINPLUS backbone and T-DNA region, and unique restriction sites were introduced to allow for accommodation of multiple expression cassettes, including that for a suppressor of silencing, on the same plasmid. These vectors allow the high-level simultaneous expression of multiple polypeptides from a single plasmid within a few days. Furthermore, vectors have been developed which allow the direct cloning of genes into the binary plasmid by both restriction enzyme-based cloning and GATEWAY recombination. In both cases, N- or C-terminal histidine tags may be fused to the target sequence as required. These vectors provide an easy and quick tool for the production of milligram quantities of recombinant proteins from plants with standard plant research techniques at a bench-top scale.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1467-7652.2009.00434.x

pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants

Acyltransferases in plants: a good time to be BAHD.

We 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. We 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. In 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.

/pdf/transient-expression-vectors-for-functional-genomics-4s6jcdwc3w.pdf

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

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.

/pdf/a-genomics-approach-reveals-that-aroma-production-in-apple-40a5acuxye.pdf

A genomics approach reveals that aroma production in apple is controlled by ethylene predominantly at the final step in each biosynthetic pathway

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 K(m) 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.

/pdf/an-alcohol-acyl-transferase-from-apple-cv-royal-gala-mpaat1-28py4y3s5q.pdf

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

Partition of fourteen volatile compounds, representing the diverse physicochemical properties of aroma compounds, was measured by static equilibrium headspace in solutions containing the components of artificial saliva, either singly or in mixtures. Comparison of a bovine salivary mucin and pig gastric mucin showed no significant difference in partition behavior of the volatiles, so gastric mucin was used. Mucin viscosity changed with pH, but binding of volatile compounds did not show a marked dependence on pH. All combinations of the salivary components were tested for their effect on partition. Three types of behavior were noted. Partition of some compounds was unaffected by mucin, and with other compounds mucin decreased partition, whereas another group showed a decrease with mucin that was affected by the presence of salivary salts and sugar. When volatiles or sugar were added to a mucin solution, the final headspace concentration depended on the order of addition, indicating some competition for binding. These solute-mucin effects are discussed in relation to mucin structure and behavior in solution.

Ellen N. Friel

Papers

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

A genomics approach reveals that aroma production in apple is controlled by ethylene predominantly at the final step in each biosynthetic pathway

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

Effect of salivary components on volatile partitioning from solutions.

An empirical model to predict the headspace concentration of volatile compounds above solutions containing sucrose