Sol Green

Multiple Repeats of a Promoter Segment Causes Transcription Factor Autoregulation in Red Apples

Mutations in the genes encoding for either the biosynthetic or transcriptional regulation of the anthocyanin pathway have been linked to color phenotypes. Generally, this is a loss of function resulting in a reduction or a change in the distribution of anthocyanin. Here, we describe a rearrangement in the upstream regulatory region of the gene encoding an apple (Malus × domestica) anthocyanin-regulating transcription factor, MYB10. We show that this modification is responsible for increasing the level of anthocyanin throughout the plant to produce a striking phenotype that includes red foliage and red fruit flesh. This rearrangement is a series of multiple repeats, forming a minisatellite-like structure that comprises five direct tandem repeats of a 23-bp sequence. This MYB10 rearrangement is present in all the red foliage apple varieties and species tested but in none of the white fleshed varieties. Transient assays demonstrated that the 23-bp sequence motif is a target of the MYB10 protein itself, and the number of repeat units correlates with an increase in transactivation by MYB10 protein. We show that the repeat motif is capable of binding MYB10 protein in electrophoretic mobility shift assays. Taken together, these results indicate that an allelic rearrangement in the promoter of MYB10 has generated an autoregulatory locus, and this autoregulation is sufficient to account for the increase in MYB10 transcript levels and subsequent ectopic accumulation of anthocyanins throughout the plant.

Two terpene synthases are responsible for the major sesquiterpenes emitted from the flowers of kiwifruit (Actinidia deliciosa)

Kiwifruit vines rely on bees for pollen transfer between spatially separated male and female individuals and require synchronized flowering to ensure pollination. Volatile terpene compounds, which are important cues for insect pollinator attraction, were studied by dynamic headspace sampling in the major green-fleshed kiwifruit (Actinidia deliciosa) cultivar ‘Hayward’ and its male pollinator ‘Chieftain’. Terpene volatile levels showed a profile dominated by the sesquiterpenes α-farnesene and germacrene D. These two compounds were emitted by all floral tissues and could be observed throughout the day, with lower levels at night. The monoterpene (E)-β-ocimene was also detected in flowers but was emitted predominantly during the day and only from petal tissue. Using a functional genomics approach, two terpene synthase (TPS) genes were isolated from a ‘Hayward’ petal EST library. Bacterial expression and transient in planta data combined with analysis by enantioselective gas chromatography revealed that one TPS produced primarily (E,E)-α-farnesene and small amounts of (E)-β-ocimene, whereas the second TPS produced primarily (+)-germacrene D. Subcellular localization using GFP fusions showed that both enzymes were localized in the cytoplasm, the site for sesquiterpene production. Real-time PCR analysis revealed that both TPS genes were expressed in the same tissues and at the same times as the corresponding floral volatiles. The results indicate that two genes can account for the major floral sesquiterpene volatiles observed in both male and female A. deliciosa flowers.

Defining the Potassium Binding Region in an Apple Terpene Synthase

Terpene synthases are a family of enzymes largely responsible for synthesizing the vast array of terpenoid compounds known to exist in nature. Formation of terpenoids from their respective 10-, 15-, or 20-carbon atom prenyl diphosphate precursors is initiated by divalent (M2+) metal ion-assisted electrophilic attack. In addition to M2+, monovalent cations (M+) have also been shown to be essential for the activity of certain terpene synthases most likely by facilitating substrate binding or catalysis. An apple α-farnesene synthase (MdAFS1), which has a dependence upon potassium (K+), was used to identify active site regions that may be important for M+ binding. Protein homology modeling revealed a surface-exposed loop (H-αl loop) in MdAFS1 that fulfilled the necessary requirements for a K+ binding region. Site-directed mutagenesis analysis of specific residues within this loop then revealed their crucial importance to this K+ response and strongly implicated specific residues in direct K+ binding. The role of the H-αl loop in terpene synthase K+ coordination was confirmed in a Conifer pinene synthase also using site-directed mutagenesis. These findings provide the first direct evidence for a specific M+ binding region in two functionally and phylogenetically divergent terpene synthases. They also provide a basis for understanding K+ activation in other terpene synthases and establish a new role for the H-αl loop region in terpene synthase catalysis.

Functional Genomics Reveals That a Compact Terpene Synthase Gene Family Can Account for Terpene Volatile Production in Apple

Summary: Apple is shown to contain only a small number of functional terpene synthase genes whose evolution appears to have been shaped by genome-wide duplication events and commercial breeding strategies. Terpenes are specialized plant metabolites that act as attractants to pollinators and as defensive compounds against pathogens and herbivores, but they also play an important role in determining the quality of horticultural food products. We show that the genome of cultivated apple (Malus domestica) contains 55 putative terpene synthase (TPS) genes, of which only 10 are predicted to be functional. This low number of predicted functional TPS genes compared with other plant species was supported by the identification of only eight potentially functional TPS enzymes in apple ‘Royal Gala’ expressed sequence tag databases, including the previously characterized apple (E,E)-α-farnesene synthase. In planta functional characterization of these TPS enzymes showed that they could account for the majority of terpene volatiles produced in cv Royal Gala, including the sesquiterpenes germacrene-D and (E)-β-caryophyllene, the monoterpenes linalool and α-pinene, and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene. Relative expression analysis of the TPS genes indicated that floral and vegetative tissues were the primary sites of terpene production in cv Royal Gala. However, production of cv Royal Gala floral-specific terpenes and TPS genes was observed in the fruit of some heritage apple cultivars. Our results suggest that the apple TPS gene family has been shaped by a combination of ancestral and more recent genome-wide duplication events. The relatively small number of functional enzymes suggests that the remaining terpenes produced in floral and vegetative and fruit tissues are maintained under a positive selective pressure, while the small number of terpenes found in the fruit of modern cultivars may be related to commercial breeding strategies.

Identification, functional characterization, and regulation of the enzyme responsible for floral (E)-nerolidol biosynthesis in kiwifruit (Actinidia chinensis)

Flowers of the kiwifruit species Actinidia chinensis produce a mixture of sesquiterpenes derived from farnesyl diphosphate (FDP) and monoterpenes derived from geranyl diphosphate (GDP). The tertiary sesquiterpene alcohol (E)-nerolidol was the major emitted volatile detected by headspace analysis. Contrastingly, in solvent extracts of the flowers, unusually high amounts of (E,E)-farnesol were observed, as well as lesser amounts of (E)-nerolidol, various farnesol and farnesal isomers, and linalool. Using a genomics-based approach, a single gene (AcNES1) was identified in an A. chinensis expressed sequence tag library that had significant homology to known floral terpene synthase enzymes. In vitro characterization of recombinant AcNES1 revealed it was an enzyme that could catalyse the conversion of FDP and GDP to the respective (E)-nerolidol and linalool terpene alcohols. Enantiomeric analysis of both AcNES1 products in vitro and floral terpenes in planta showed that (S)-(E)-nerolidol was the predominant enantiomer. Real-time PCR analysis indicated peak expression of AcNES1 correlated with peak (E)-nerolidol, but not linalool accumulation in flowers. This result, together with subcellular protein localization to the cytoplasm, indicated that AcNES1 was acting as a (S)-(E)-nerolidol synthase in A. chinensis flowers. The synthesis of high (E,E)-farnesol levels appears to compete for the available pool of FDP utilized by AcNES1 for sesquiterpene biosynthesis and hence strongly influences the accumulation and emission of (E)-nerolidol in A. chinensis flowers.

Predicting the substrate specificity of a glycosyltransferase implicated in the production of phenolic volatiles in tomato fruit

The volatile compounds that constitute the fruit aroma of ripe tomato (Solanum lycopersicum) are often sequestered in glycosylated form. A homology‐based screen was used to identify the gene SlUGT5, which is a member of UDP‐glycosyltransferase 72 family and shows specificity towards a range of substrates, including flavonoid, flavanols, hydroquinone, xenobiotics and chlorinated pollutants. SlUGT5 was shown to be expressed primarily in ripening fruit and flowers, and mapped to chromosome I in a region containing a QTL that affected the content of guaiacol and eugenol in tomato crosses. Recombinant SlUGT5 protein demonstrated significant activity towards guaiacol and eugenol, as well as benzyl alcohol and methyl salicylate; however, the highest in vitro activity and affinity was shown for hydroquinone and salicyl alcohol. NMR analysis identified isosalicin as the only product of salicyl alcohol glycosylation. Protein modelling and substrate docking analysis were used to assess the basis for the substrate specificity of SlUGT5. The analysis correctly predicted the interactions with SlUGT5 substrates, and also indicated that increased hydrogen bonding, due to the presence of a second hydrophilic group in methyl salicylate, guaiacol and hydroquinone, appeared to more favourably anchor these acceptors within the glycosylation site, leading to increased stability, higher activities and higher substrate affinities.

Natural Variation in Monoterpene Synthesis in Kiwifruit: Transcriptional Regulation of Terpene Synthases by NAC and ETHYLENE-INSENSITIVE3-Like Transcription Factors

Fruit monoterpene synthesis in kiwifruit is transcriptionally regulated by transcription factors that activate the terpene synthase promoter. Two kiwifruit (Actinidia) species with contrasting terpene profiles were compared to understand the regulation of fruit monoterpene production. High rates of terpinolene production in ripe Actinidia arguta fruit were correlated with increasing gene and protein expression of A. arguta terpene synthase1 (AaTPS1) and correlated with an increase in transcript levels of the 2-C-methyl-d-erythritol 4-phosphate pathway enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXS). Actinidia chinensis terpene synthase1 (AcTPS1) was identified as part of an array of eight tandemly duplicated genes, and AcTPS1 expression and terpene production were observed only at low levels in developing fruit. Transient overexpression of DXS in Nicotiana benthamiana leaves elevated monoterpene synthesis by AaTPS1 more than 100-fold, indicating that DXS is likely to be the key step in regulating 2-C-methyl-d-erythritol 4-phosphate substrate flux in kiwifruit. Comparative promoter analysis identified potential NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor) and ETHYLENE-INSENSITIVE3-like transcription factor (TF) binding sites in the AaTPS1 promoter, and cloned members of both TF classes were able to activate the AaTPS1 promoter in transient assays. Electrophoretic mobility shift assays showed that AaNAC2, AaNAC3, and AaNAC4 bind a 28-bp fragment of the proximal NAC binding site in the AaTPS1 promoter but not the A. chinensis AcTPS1 promoter, where the NAC binding site was mutated. Activation could be restored by reintroducing multiple repeats of the 12-bp NAC core-binding motif. The absence of NAC transcriptional activation in ripe A. chinensis fruit can account for the low accumulation of AcTPS1 transcript, protein, and monoterpene volatiles in this species. These results indicate the importance of NAC TFs in controlling monoterpene production and other traits in ripening fruits.

Mapping, Complementation, and Targets of the Cysteine Protease Actinidin in Kiwifruit

Cysteine proteases (CPs) accumulate to high concentration in many fruit, where they are believed to play a role in fungal and insect defense. The fruit of Actinidia species (kiwifruit) exhibit a range of CP activities (e.g. the Actinidia chinensis variety YellowA shows less than 2% of the activity of Actinidia deliciosa variety Hayward). A major quantitative trait locus for CP activity was mapped to linkage group 16 in a segregating population of A. chinensis. This quantitative trait locus colocated with the gene encoding actinidin, the major acidic CP in ripe Hayward fruit encoded by the ACT1A-1 allele. Sequence analysis indicated that the ACT1A locus in the segregating A. chinensis population contained one functional allele (A-2) and three nonfunctional alleles (a-3, a-4, and a-5) each containing a unique frameshift mutation. YellowA kiwifruit contained two further alleles: a-6, which was nonfunctional because of a large insertion, and a-7, which produced an inactive enzyme. Site-directed mutagenesis of the act1a-7 protein revealed a residue that restored CP activity. Expression of the functional ACT1A-1 cDNA in transgenic plants complemented the natural YellowA mutations and partially restored CP activity in fruit. Two consequences of the increase in CP activity were enhanced degradation of gelatin-based jellies in vitro and an increase in the processing of a class IV chitinase in planta. These results provide new insight into key residues required for CP activity and the in vivo protein targets of actinidin.

In planta transient expression analysis of monoterpene synthases.

In vitro-based analyses of monoterpene synthase (mono-TPS) enzymes have led to a wealth of knowledge regarding their catalytic behavior, the mechanistic principles governing their product specificity, and the molecular basis for their evolution. However, the efficient production of active enzymes in Escherichia coli or yeast can be challenging. Agrobacterium-mediated transient expression in tobacco leaves is increasingly being used as a viable alternative to in vitro-based approaches for the production and functional analysis of a wide range of plant proteins. Transient expression is well suited for qualitative and semiquantitative analyses of mono-TPS enzyme product specificity and, in conjunction with standard volatile analysis techniques, provides an efficient tool for screening mono-TPS function in planta. The primary advantages of this system for mono-TPS analysis are that both mono-TPS genomic clones and cDNAs can be cloned directly into plant expression vectors without modification and expressed enzymes can be analyzed without the need for purification or endogenous precursor addition. Here, we describe a simple and cost-effective method for the in planta functional analysis of plant mono-TPS enzymes. This method can accommodate both the analysis of single genes and the scaling for more high-throughput functional screening of mono-TPS gene families or mutant libraries.

A non-synonymous nucleotide substitution can account for one evolutionary route to sesquiterpene synthase activity in the TPS-b subgroup

Plant sesquiterpene and hemiterpene synthases in the monoterpene synthase dominated TPS‐b subgroup are thought to have evolved independently from a monoterpene synthase ancestor. A TPS‐b sesquiterpene synthase from apple (MdAFS1), which predominantly produces α‐farnesene, can also synthesize the monoterpene (E)‐β‐ocimene. The dual activity offered a functional link to an ancestral MdAFS1 enzyme and a rational basis for investigation of the evolution of TPS‐b sesquiterpene enzymes. Protein modelling and mutagenesis analysis of the MdAFS1 active site identified a non‐synonymous nucleotide substitution that could account for the requisite shift in substrate specificity necessary for the emergence of its sesquiterpene activity during the evolution of the TPS‐b enzymes.