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More than meets the noseRegulation of floral scent biosynthesis in PetuniaBoersma, M.R.

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Citation for published version (APA):Boersma, M. R. (2018). More than meets the nose: Regulation of floral scent biosynthesis in Petunia.

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Download date: 16 Jun 2020

CHAPTER 1

INTRODUCTION

Floral scent production by Petunia in the genome era

Maaike R. Boersma,a Joëlle K. Muhlemann,b1 Avichai Amrad,c Natalia Dudareva,b Cris Kuhlemeier,c Michel A. Haring,a and Robert C. Schuurinka2

a Department of Plant Physiology, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands

b Department of Biochemistry, Purdue University, 47907-2063 West Lafayette, IN, USA

c Institute of Plant Sciences, University of Bern, Altenbergrain 21, 3013 Bern, Switzerland

1 Current address: Department of Biology, Wake Forest University, 27109-1834 Winston-Salem, NC, USA

2 Address correspondences to R.C.Schuurink@uva.nl

This chapter has been published as: Supplementary note 8: M.R. Boersma, J.K. Muhlemann, A. Amrad, N. Dudareva C. Kuhlemeier, M.A. Haring, and R.C. Schuurink ‘Extreme variation in volatile production in wild petunias: what can we learn from their genomes?’ of Bombarely, A et al. Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida (2016) Nature plants 2, 16074

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Petunia as a model system to study (floral volatile) benzenoid and phenylpropanoid biosynthesis

Pollination by insects is dependent on floral scent in many plant species 1-3. The abundance, ratio and composition of the volatiles in the floral headspace are suggested to be involved in reproductive isolation of closely related species 4. The composition of the floral headspace is shaped by the selective pressures exerted by both pollinators and florivores 5. Within the Petunia floral headspace, certain volatiles were shown to attract pollinators, while others deter florivores 6. Although little is known about the function of individual volatiles and specific volatile mixes on insect behavior, Petunia species display either moth or bee-pollinated phenotypes. For example, P. axillaris flowers are white and scented, which is characteristic for nocturnal hawkmoth pollination 1,7 whereas P. integrifolia has purple flowers which make a few volatiles and are pollinated by bees 7,8. Scent is an important cue for hawk-moth visitation of Petunia flowers 1, and the emission of volatiles is timed with pollinator activity 9. Besides volatile emission being regulated diurnally in Petunia, emission of volatiles is also petal-specific and developmentally regulated. Emission of volatiles starts in open flowers at anther dehiscence and lasts until flower senescence 9. This tight regulation of volatile emission and the variation of volatile emission in both wild and hybrid Petunias has made Petunia the model system for floral volatile studies. With the recent publication of the genomes of the ancestors of the Petunia hybrida collection, P.axillaris axillaris N (PaxiN) and P. integrifolia inflata S6 (PinfS6), the evolutionary changes causing differences in emission of volatiles can be studied 10.

Petunia floral scent is mainly composed of phenylpropanoids and benzenoids 9, which are the second most abundant and widespread class of plant volatiles 11 (Figure 1). Besides playing an important role in pollination of flowering plants benzenoids and phenylpropanoids are the precursors for many important compounds in plants. For example, benzenoid precursors and benzoic acid are the precursors for the hormone salicylic acid 12, and phenylpropanoids are the precursors for lignin, spermidines, flavonoids and anthocyanins 13. Petunia has proven itself as a valuable model system to study benzenoid and phenylpropanoid biosynthesis. Knowledge on phenylpropanoid and benzenoid biosynthesis from Petunia could be used to study and improve the taste and smell of economically important crops such as basil and tomato.

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Figure 1. Biosynthesis of floral volatile benzenoids and phenylpropanoids by Petunia. Floral volatiles emitted by Petunia are red. Biosynthetic genes and steps in grey have not been characterized in Petunia. Abbreviations: DAHPS: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase; DHQS: 3-dehydroquinate synthase; SD: shikimate dehydrogenase; SK: shikimate kinase: EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase: CS: chorismate synthase; ADT: arogenate dehydratase; PPA-AT: prephenate aminotransferase; PPY-AT: phenylpyruvate aminotransferase; CM: chorismate mutase; PAL: L-phenylalanine ammonia lyase; BPBT: benzoyl-CoA:benzylalcohol/2-phenylethanol benzoyltransferase; PAAS: phenylacetaldehyde synthase; CNL: cinnamate-CoA ligase; CHD: cinnamoyl-CoA hydratase-dehydrogenase; KAT: 3-ketoacyl-CoA thiolase; BALDH: benzaldehyde dehydrogenase; BSMT: S-adenosyl-L-methionine:benzoic acid/salicylic acid carboxyl methyltransferase; C4H: cinnamate 4-hydoxylase; 4CL: 4-coumarate CoA-ligase; HCT: hydroxycinnamoyl-transferase; CCoAOMT: caffeoyl-CoA 3-O-methyltransferase; CCR: cinnamoyl-CoA reductase; CAD: cinnamyl alcohol dehydrogenase; CFAT: coniferylalcohol acetyl-CoA:coniferyl alcohol acetyltransferase; EGS: eugenol synthase; IGS: isoeugenol synthase; C6-Cx: benzenoid and phenylpropanoid compounds with one, two or three carbon side chain attached to the benzene ring.

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Biosynthesis of phenylpropanoids and benzenoids by Petunia

Floral volatile benzenoids and phenylpropanoids (FVBPs) are derived from the precursor L-phenylalanine (L-Phe), which originates from the shikimate and arogenate pathway (Figure 1) 14-16. The phenylpropanoid related (C6-C2) compounds phenylethanol, phenylacetaldehyde and phenylethylacetate are synthesized via an unusual pathway in Petunia, in which phenylacetaldehyde is directly produced from L-Phe by phenylacetaldehyde synthase (PAAS) 17. The biosynthesis of both benzenoids (C6-C1) and phenylpropanoids (C6-C3) starts with the deamination of L-Phe to trans-cinnamic acid (t-CA) by L-phenylalanine ammonia lyase (PAL). The C6-C1 pathway proceeds through the β-oxidative and non-β-oxidative pathway and yields benzylalcohol, methylbenzoate (MeBA), benzylacetate, methylsalicylate (MeSA) and benzylbenzoate 18-22. Phenylethylbenzoate can be derived from both the C6-C1 as the C6-C2 pathway. Cinnamate 4-hydoxylase (C4H) catalyzes the first committed step to C6-C3 biosynthesis, but also provides precursors for lignin, spermidines, flavonoids and anthocyanins biosynthesis. The C6-C3 volatiles isoeugenol and eugenol share the precursor coniferylalcohol with the lignin pathway 23, whose oxygen group is eliminated during isoeugenol and eugenol formation 24. We have identified cinnamyl alcohol dehydrogenase (CAD) in Petunia, an enzyme that catalyzes the conversion of cinnamyl alcohol to cinnamaldehyde (CHAPTER 3). The exact biosynthetic route leading to vanillin and benzaldehyde is still unknown (Figure 1). It has been suggested that benzaldehyde is an important intermediate for benzoic acid (BA) biosynthesis. However t-CA, BA and salicylic acid, but not benzaldehyde, were labeled when Petunia was fed with radiolabelled L-Phe 25,26. Moreover, it has been hypothesized that benzaldehyde is derived from BA and not the other way around 27. Transcriptional regulation of scent biosynthesis and emission

Biosynthesis of FVBPs is intricately regulated by an array of R2R3-MYB transcription factors in Petunia (Table 1). The R2R3-MYB transcription factors ODORANT1 (ODO1), EMISION OF BENZENOIDSI/II/V (EOBI/II/V) and MYB4 are known to play a role in regulating FVBPs biosynthesis 32-36. Silencing of ODO1 (ODO-ir) in P. hybrida cv. W115 results in reduced emission and storage of most FVBPs. ODO1 regulates the precursor supply to the FVBP pathway by activating the promoter of the shikimate enzyme 5-enolpyruvylshikimate-3-phosphatse synthase (EPSPS) 31. RNA-sequencing of an ODO1-ir line revealed that many genes in the shikimate and arogenate pathway are regulated by ODO1 (CHAPTER 3). In addition, the expression of all C6-C3 biosynthetic genes except isoeugenol synthase (IGS) and eugenol synthase (EGS) are reduced upon silencing of ODO1. Chromatin immunoprecipitation followed

INTRODUCTION

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by sequencing (ChIP-seq) of GFP tagged ODO1 showed that some of these genes are directly regulated by ODO1 (CHAPTER 3).

ODO1 thus plays a crucial role in regulating FVBPs biosynthesis and is in a feedback loop with two other positive regulators of FVBP biosynthesis, EOBI and EOBII. ODO1 is expressed in the evening before nocturnal FVBP emission and is activated by midday-phase expressed EOBI and EOBII. In turn ODO1 negatively regulates EOBI expression (Figure 2) 32-34,37. Although EOBII can activate the ODO1 promoter, additional factors are required for full activation of the ODO1 promoter 37. EOBI and EOBII are very similar proteins and have been shown to both bind the promoters of PAL and IGS. However, silencing of these transcription factors does not lead to an identical reduction in emission of volatiles. For example, benzaldehyde emission is reduced upon silencing of EOBII, but not upon silencing of EOBI 29,30 (Table 1). MYB4 and EOBV are both negative regulators of FVBP biosynthesis. MYB4 negatively regulates C4H and thereby eugenol and isoeugenol biosynthesis. Emission of the C6-C2

and C6-C1 volatiles phenylacteladehyde and MeBA is reduced upon silencing of MYB4 whereas eugenol and isoeugenol emission increases 35. The exact role of EOBV is not yet clear, although a role as a negative regulator has recently been suggested. Under increased ambient temperatures volatile emission is reduced and EOBV expression increased. In addition, silencing of EOBV leads to increased volatile emission 38. Recently a regulator of the circadian clock and a regulator of anthocyanin biosynthesis have been described to play a role in FVBP emission. The clock gene LATE ELONGATED

Figure 2. Transcriptional regulation of floral volatile benzenoids and phenylpropanoids biosynthesis 28-31. A transcriptional network regulates biosynthesis of floral volatiles in Petunia by binding to promoters of biosynthetic genes. Transcription factors are circled and activation or inhibition of genes is depicted with arrows and blunt arrows, respectively (a). Circadian gene expression levels of LHY, EOBI, EOBII and ODO1 are depicted schematically. The white and black bars indicate the light and dark period, respectively (b). Abbreviations: LHY: LATE ELONGATED HYPOCOTYL; ODO1: ODORANT1; EOBI&II&V: EMMISION OF BENZENOIDSI&II&V; EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase; ABCG1: ATP-binding cassette transporter G1; PAL: L-phenylalanine ammonia lyase; IGS: isoeugenol synthase, C4H: cinnamate 4-hydoxylase.

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Table 1. Overview of the effect of silencing or overexpressing transcription factors on emission of floral volatiles 28-31,34. Abbreviations: LHY: LATE ELONGATED HYPOCOTYL; ODO1: ODORANT1; EOB: EMMISION OF BENZENOIDS; MYB: myeloblastosis; FVBP: floral volatile benzenoids and phenylpropanoids; C6-Cx: benzenoid and phenylpropanoid compounds with one, two or three carbon side chain attached to the benzene ring. ↑ LHY

↓ C6-C1 ↓ C6-C3

benzylbenzoate isoeugenol

benzaldehyde

methyl benzoate

↓ ODO1 ↓C6-C2 ↓ C6-C1 ↓ C6-C3 phenylacetaldehyde benzylbenzoate vanillin phenylethanol methylbenzoate isoeugenol benzylacetate

↓ EOBI ↓C6-C2 ↓ C6-C1 ↓ C6-C3 phenylacetaldehyde benzylbenzoate isoeugenol phenylethylbenzoate benzylalcohol eugenol

methyl salicylate

methyl benzoate

↓ EOBII ↓C6-C2 ↓ C6-C1 ↓ C6-C3 phenylethanol benzylbenzoate vanillin phenylethylacetate benzaldehyde isoeugenol

benzylalcohol eugenol

benzylacetate

↓ MYB4 ↓C6-C2 ↓ C6-C1 ↑ C6-C3 phenylacetaldehyde methyl benzoate isoeugenol eugenol

↓ EOBV ↑ total pool of FVBP's

↓ PH4 ↓C6-C2 ↓ C6-C1 ↓ C6-C3 phenylacetaldehyde benzylbenzoate vanillin phenylethanol benzaldehyde isoeugenol

benzylalcohol eugenol

benzylacetate

INTRODUCTION

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HYPOCOTYL (LHY) seems to directly determine the diurnal rhythm of FVBP production by negatively regulating ODO1 expression. LHY is expressed high in the morning when ODO1 expression is low (Figure 2). Increased expression of LHY and silencing of ODO1 leads to reduced emission of the same volatiles (Table 1) 28. Emission of volatiles has recently been shown to be regulated by the R2R3-MYB transcription factor PH4 39, which is predominantly known for its role in vacuolar acidification in relation to anthocyanin biosynthesis 40. Virus induced genes silencing (VIGS) of PH4 led to decreased emission, but increased accumulation of FVBPs 41. The role of transport in scent biosynthesis and emission

Besides transcription factors also transporters regulate FVBP accumulation and emission. Recently a plastidial cationic amino-acid transporter (PhpCAT) has been described to play a role in FVBP production by Petunia. PhpCAT exports L-Phe, the precursor of FVBPs, as well as tyrosine and tryptophan, from the plastid. Silencing of PhpCAT leads to reduced pools of L-Phe and tyrosine and reduced emission of FVBPs 42. The localization of the shikimate pathway in the plastid does not only require export of its products, but also import of its precursors phosphoenol pyruvate (PEP) and erythrose-4-phosphate (E4P). We have discovered a putative phosphoenolpyruvate transporter (PEPT) (CHAPTER 3). The discovery of this plastidial transporter is only the start of understanding the role of transport in FVBP biosynthesis. Besides playing a role in regulating of FVBP production, compartmentalization of phenylpropanoid biosynthesis has a more fundamental function. Compartmentalization of FVBP biosynthesis and formation of enzyme complexes can protect the cell from toxic phenylpropanoid intermediates 43. Both acyl-activating enzyme (AAE, also known as CNL) and KAT1 are peroxisomal localized in Petunia indicating compartmentalization of benzoyl-CoA biosynthesis 20,44. Another example of co-localization and potential enzymatic complex formation was observed in tobacco. PAL1 co-localizes with C4H on the ER membrane, whereas PAL2 localization changes from cytosolic to the ER membrane upon overexpression of C4H 45.

Finally, volatiles need to be released from cells. For a long time it was assumed that emission of volatiles mainly occurs by diffusion. However, a recent modeling study shows that the non-polar character of most volatiles will lead to extreme high concentration in membranes. Therefore active mechanisms for the release of volatiles are required to prevent disturbance of membrane integrity 46. A candidate for FVBP emission in Petunia is the plasma-membrane localized ABCG1 transporter. The Arabidopsis homolog AtABCG29 transports p-coumaryl alcohol, a compound structurally related to (iso)eugenol and vanillin 23,46,47. Moreover, the key regulator of FVBP emission, ODO1, activates the promoter of ABCG1. Recently it has been shown that ABCG1 indeed is involved in emission of FVBP by means of active transport 48.

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Interestingly, a localization study in Petunia petal protoplasts showed that ABCG1, ODO1 and IGS promoters are active in both mesophyll as epidermal cells 49. This suggests that FVBP emission does not only require trafficking between cellular compartments and emission from epidermal cells, but also trafficking from the mesophyll to the epidermis since guard cells/stomata are scarcely present in petals. Differences in scent biosynthesis between P. axillaris axillaris N (PaxiN) and P. integrifolia inflata S6 (PinfS6)

The difference in floral scent production between closely related species of Petunia makes this an excellent example to study evolutionary changes underlying shifts in FVBP biosynthesis 5. A recent phylogenetic analysis of the genus robustly puts P. integrifolia and related bee-pollinated species into the ancestral short-tube clade, from which long-tubed species such P. axillaris are derived 50. Therefore, the evolution of a hawkmoth-pollination syndrome from a bee-pollinated ancestor has involved major changes in the amounts, complexity and diurnal regulation of FVBPs. The availability of high quality genome sequences of representatives of the two clades, P. axillaris axillaris N (PaxiN) and P. integrifolia inflata S6 (PinfS6), provides new tools to study the genetic and genomic basis of this evolutionary transition.

PinfS6 emits high levels of benzaldehyde 51-53 and low levels of isoeugenol and eugenol (CHAPTER 2). This means that PaxiN has gained the ability to emit a more abundant and complex blend of volatiles, existing of benzylbenzoate, phenylacetaldehyde, methylbenzoate (MeBA), benzaldehyde, phenylethylbenzoate, isoeugenol 49,51 and eugenol (CHAPTER 2). To investigate whether divergence of genes underlies the difference in complexity of the floral blend between PinfS6 and PaxiN, the coding sequences (CDS) of FVBP genes were annotated and compared after translation 10. One can predict that an (early) stop codon or a frame shift in the CDS of a biosynthetic gene will cause the loss of a volatile from the floral headspace. This was observed for IGS and isoeugenol synthesis in Petunia axillaris subsp. parodii 54. Thus, all known biosynthetic genes, i.e. those involved in benzylbenzoate, phenylacetaldehyde, MeBA, phenylethylbenzoate, isoeugenol and eugenol biosynthesis were investigated (Suppl. table 1&2) 10. The analysis of both genomes shows that there are neither frame shifts nor stop codons present in the PinfS6 FVBP biosynthetic genes that have been compared to PaxiN. Moreover, the PinfS6 and PaxiN FVBP biosynthetic proteins analyzed differ only 2% on average, and most of those changes are silent or conservative (Suppl. table 1&2) 10. Small functional differences caused by changes in the protein sequences cannot be excluded, but it seems unlikely that these can account for the massive and complex increase of volatile emission in P. axillaris. Conversely, the lack of FVBP biosynthesis in PinfS6 could be the result of a mutation in upstream biosynthetic genes preventing precursor supply to the FVBP pathway. L-Phe

INTRODUCTION

13

is an essential constituent of proteins as well as the precursor for many different secondary metabolites 13. Functional mutation of this pathway would probably lead to serious defects in plants, and this could explain the presence of multiple copies of shikimate and arogenate pathway genes and PAL genes in plants. Nevertheless, there are examples in which silencing of specific shikimate and arogenate pathway genes (CM1, DAPHS1 and ADT1) leads to reduced FVBP emission in Petunia without causing severe phenotypes 16,32,55,56. In addition, the functionality of the known L-Phe biosynthetic genes CM1, DAPHS1 and ADT1, is likely not altered in PinfS6. CM1, DAPHS1 and ADT1a/ADT1b have only three, five and three amino acid changes compared to PaxiN, respectively 10. Although the amino acid sequence of ADT1b is identical to ADT1a in PinfS6, it contains an intron unlike ADT1a.

The relatively few quantitative trait loci (QTL) identified by QTL analysis 1,53 indicate the presence of major functional differences in yet unidentified genes or in the promoters of genes analyzed. However, it cannot be excluded that changes in the protein sequences collectively underlie the phenotypic differences as polygenic trait as argued by Rockman 57. Recently it has been shown that the biosynthetic genes S-adenosyl-L-methionine:benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) and benzoyl-CoA:benzylalcohol/2-phenylethanol benzoyltransferase (BPBT) are not expressed in PinfS6, but are in PaxiN 58. Gain of BSMT and BPBT expression can explain the gain of phenylethylbenzoate, benzylbenzoate and methylbenzoate production by PaxiN 58. An F1 from a PaxiN and PinfS6 cross emits methylbenzoate and emits lower levels of benzaldehyde than PinfS6. RNA-seq and allele-specific expression (ASE) analysis of this F1 PaxiN and PinfS6 cross showed that BSMT1, BSMT2 and BPBT2 are PaxiN allele specific. This indicates that the promoter regions of BSMT and BPBT in PinfS6 are not functional. However, methylbenzoate emission of this F1 cross is only 10% of the methylbenzoate emission of PaxiN. Additional factors, that are absent in PinfS6, are required for higher levels of methylbenzoate emission 58. Transcriptional regulation of scent biosynthesis in PaxiN and PinfS6

The single-gene hypothesis can explain fast adaptations: the rapid adaption of floral traits can be controlled by mutation of a single regulator 59. For instance, corolla color is under control of such a single regulator. The loss of color in Petunia is explained by mutations in the R2R3 MYB transcription factor ANTHOCYANIN2 (AN2) 52. Therefore it was investigated whether the difference in volatile emission by PaxiN and PinfS6 can be explained by genetic variation in key transcriptional regulators of FVBP biosynthesis and emission. Prior evidence points to such a role for ODO1. Mutations of the EOBII binding sites in the ODO1 promoter are sufficient to lose the ability to synthesize FVBPs 34. Moreover, two QTLs explain the difference in FVBP biosynthesis in

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PaxiN and P. exserta, and ODO1 is located in one of these two QTLs on chromosome VII 1. It is expected that a lower level of ODO1 expression would result in lower levels of all FVBPs. However, ODO1 is expressed in both PaxiN and PinfS6 to a similar extend (CHAPTER 2). Further analysis of ODO1 revealed that there are three non-conserved amino acid substitutions in PinfS6 compared to the fragrant PaxiN and P. hybrida cv. W115 (Figure 3). It remains to be determined if these mutations have consequences for the activity of ODO1 in PinfS6. Interestingly, P. hybrida cv. W115 has five amino acid changes that are similar to PaxiN and two amino acid changes that are similar to PinfS6 (Figure 3). Of the 20,000 genes investigated in the P. hybrida lines W115, R27 and R143, about 15,000 genes are derived from PaxiN and 600 genes are derived from PinfS6. In addition 1,500 genes seem to be from mixed origin as observed for ODO1. The reasons for these mixed genes could be the involvement of an unknown parent besides PaxiN and PinfS6, gene conversions or random repair of heteroduplexes 10. Interestingly, silencing of one of the other FVBP regulators, EOBI, in P. hybrida cv. W115 does not alter benzaldehyde emission. Whereas all other volatiles that are also emitted by PaxiN are affected by EOBI silencing 29. It suggests that benzaldehyde emission could be regulated differently than emission of other FVBPs. EOBII and EOBI are more conserved than ODO1 when comparing PinfS6 and PaxiN: both have only one amino acid substitution. This amino acid change is conservative in EOBII. In addition,

Figure 3. Amino acid sequence alignment of Petunia ODO1. ODORANT1 (ODO1) from P.axillaris axillaris N (PaxiN), P. integrifolia inflata S6 (PinfS6) and P. hybrida cv. Mitchell (W115) were aligned with ClustalW2. Yellow indicates amino acids changes that are conserved between PaxiN and PinfS6 (Gonnet PAM 250 scoring >250), these amino acids are bold if they are similar between PinfS6 and W115. Red indicates amino acids that are not conserved between PaxiN and PinfS6, but are similar between PaxiN and W115.

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the PinfS6 genome harbors EOBIb, which has eight amino acid changes and one deletion compared to EOBIa. Altogether there seems to be no differences in the coding sequences of EOBI and EOBII between PaxiN and PinfS6 that can explain the difference in FVBP biosynthesis 60,61. Recently a role for PH4, a vacuolar acidification regulator, in FVBP emission has been proposed. Even though the mechanism by which PH4 regulates volatile emission remains elusive, it is unlikely that PH4 causes the differences in volatile emission between PaxiN and PinfS6. Both PaxiN and PinfS6 carry a functional allele for PH4 10. The presence of a negative regulator in PinfS6, down regulating biosynthesis of benzylbenzoate, 2-phenylacetaldehyde, MeBA, phenylethylbenzoate and isoeugenol, could explain the differences between PinfS6 and PaxiN. The only two known negative regulators of FVBP biosynthesis, besides PH4, are MYB4 and EOBV. MYB4 specifically regulates isoeugenol and eugenol biosynthesis 35. The exact role of EOBV is still unknown, but silencing of EOBV leads to increased overall volatile emission, that is not further specified 33. Recently a role as a wide-range transcriptional repressor of the pathway was suggested for EOBV 38. EOBV has four amino acid changes and a deletion of six amino acids in PaxiN compared to PinfS6. Since activity data of EOBV are not available, the potential effect of mutations in EOBV on FVBP biosynthesis remains elusive.

FVBP emission by PaxiN and PinfS6 do not only differ in abundance and complexity, but also in timing. Whereas emission of FVBP by PaxiN starts at dusk and is timed with hawk-moth activity, FVBP emission by the bee-pollinated PinfS6 peaks during the day (CHAPTER 2). Recently, the circadian clock gene LHY has been shown to regulate rhythmic FVBP emission by binding to the promotor of ODO1, PAL1 and IGS (Figure 2 and 3)28. Both Petunia species have only one copy of LHY. Overall evening loop genes have more paralogues in PinfS6 than in PaxiN. And within the Solanaceae the circadian clock genes appear to be species specific 10. Striking differences in benzenoid and phenylpropanoid biosynthesis by Petunia compared to other sequenced Solanaceae

Petunia is part of the South America originated Solanaceae family. The genus Petunia is an early branching, separate clade with in general a larger genome (estimated size is 1.4Gb) divided over less chromosomes (x=7) 10. Interestingly some parts of the benzenoid and phenylpropanoid pathway seem to have evolved differently between these species. For example, Petunia phenylacetaldehyde is synthesized by the enzyme PAAS, whereas tomato requires two enzymatic steps for the conversion of L-Phe 17,62. Petunia PAAS genes cluster in a clade separate from putative N. benthamiana, S. lycopersicum and S. tuberosum PAAS genes (Figure 4a). The tomato gene most homologous to PaxiNPAAS2, encodes a protein that is 124

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amino acid shorter than its Petunia homolog. This might implicate that PAAS function has been lost in tomato and two other genes evolved in tomato to catalyze phenylacetaldehyde formation.

Anther interesting evolutionary event appears to have occurred for (iso)eugenol biosynthesis. IGS from Petunia seems to be more different from other Solanaceae genes than Petunia EGS. Tomato is not known to produce isoeugenol, and the lack of IGS genes in N. benthamiana, S. lycopersicum and S. tuberosum genomes implies that IGS has been lost or gained in different Solanaceae genera (Figure 4b). Moreover, a study comparing IGS and EGS between Ocimum basilicum, Clarkia breweri and P. hybrida led to the hypothesis that EGS and IGS have evolved independently at least twice in different plant lineages 63. The Petunia biosynthetic gene directly upstream of IGS, CFAT, also falls in a separate clade compared to genes from N. benthamiana, S. lycopersicum and S. tuberosum (Figure 4c). Although Petunia EGS has homologous genes in other Solanaceae (Figure 4b), and tomato has been reported to synthesize eugenol 64, genera within the Solanaceae probably have a different acyltransferase responsible for the conversion of coniferyl alcohol. The presence of a limited number of FVBP-producing species in the Solanaceae makes it difficult to unravel how benzenoid/phenylpropanoid biosynthesis has evolved in this family. However, these results clearly show that the Solanaceae have evolved multiply strategies for (iso)eugenol and 2-phenylacetaldehyde biosynthesis.

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Figure 4. Unrooted neighbor joining trees of PAAS (a), IGS and EGS (b) and CFAT (c). Unrooted neighbor joining trees from the protein sequence of different Solanaceae were created with MEGA6.06. Numbers represent Bootstrap values for 1000 replicates. Petunia identifiers are listed in supplemental table 1. Abbreviations: PAAS: phenylacetaldehyde synthase; CFAT: coniferylalcohol acetyl-CoA:coniferyl alcohol acetyltransferase; IGS: isoeugenol synthase; EGS: eugenol synthase; PaxiN: Petunia axillaris axillaris N; PinfS6: Petunia integrifolia infalata S6; Solyc: Solanum lycopersicum; Sotub: Solanum tuberesum; Nb: Nicotiana benthamiana. Thesis outline

The aim of this thesis is to unravel the transcriptional regulation of the FVBP pathway in Petunia. Although many transcription factors have been identified these are not sufficient to explain the tight developmental, rhythmic and tissue specific

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regulation of FVBP biosynthesis and emission. For example, which additional factor is required for full activation of the ODO1 promoter besides EOBII? Moreover, the current knowledge cannot explain the wide diversity observed in both wild Petunia species and Petunia hybrids in timing, abundance and complexity of volatile emission. For instance, which yet unidentified gene(s) or mechanism is responsible for the difference between PaxiN and PinfS6?

In CHAPTER 2 we start with a thorough study of the differences in volatile emission and storage between PaxiN and PinfS6. In this study we have identified additional volatiles emitted by PaxiN and PinfS6. Subsequently we have used RNA-seq data of PaxiN and PinfS6 to identify differently expressed FVBP genes. Interestingly, ODO1 expression is similar in PaxiN and PinfS6, meaning another regulator or mechanism is responsible for the overall decrease in expression of FVBP genes and reduced emission of FVBPs. Differentially expression of MYB4 and EOBII might explain some of the differences between PaxiN and PinfS6.

CHAPTER 3 describes the identification of (in)direct target genes of ODO1. The combined data of ChIP-seq experiments with GFP tagged ODO1 and RNA-seq of an ODO1-silencing line show that a big set of FVBP genes are regulated by ODO1. Most shikimate, arogenate and C6-C3 biosynthetic genes are expressed at lower levels upon silencing of ODO1. Moreover, new target genes of ODO1, which are putatively involved in FVBP biosynthesis, have been identified. Among these are two CINNAMYL ALCOHOL DEHYDROGENASES (CAD), an enzyme in the C6-C3 pathway leading to ioseugenol and eugenol biosynthesis. Research on FVBP biosynthesis has been mainly focused at the RNA and DNA level. But a large-scale study in Arabidopsis showed that although many genes display diurnal expression patterns only few (key) proteins encode by these genes display the same cycling pattern 65. In CHAPTER 4 we show that ODO1 displays rhythmic cycling at the protein level as has been suggested to be the case for key regulators. In addition this chapter shows the localization of ODO1 and posttranslational modification of ODO1. These data suggest a strict regulation of ODO1 at the protein level. In CHAPTER 5 yeast-2-hybrid screens have been performed to find interacting partners of ODO1 and EOBI, which are required for strong activation of their target genes. Y2H screens with ODO1 resulted in the discovery of two uncharacterized proteins. EOBII interacts with defense related proteins and S-adenosylmethionine synthase. CHAPTER 6 discusses the results of this thesis.

INTRODUCTION

19

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56 Colquhoun, T. A. et al. A petunia chorismate mutase specialized for the production of floral volatiles. The Plant Journal 61, 145-155, doi:10.1111/j.1365-313X.2009.04042.x (2010).

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58 Amrad, A. et al. Gain and Loss of Floral Scent Production through Changes in Structural Genes during Pollinator-Mediated Speciation. Curr Biol 26, 3303-3312, doi:10.1016/j.cub.2016.10.023 (2016).

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60 Tohge, T., Watanabe, M., Hoefgen, R. & Fernie, A. R. The evolution of phenylpropanoid metabolism in the green lineage. Crit Rev Biochem Mol Biol 48, 123-152, doi:10.3109/10409238.2012.758083 (2013).

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INTRODUCTION

23

Supplemental table 1. GeneIDs of PaxiN and PinfS6 FVBP genes. See figure 1 and table 1 for abbreviations. Paxin genome v162 and annotation v4, and PinfS6 genome v1.0.1 and annotation v3.

New genes PaxiN geneID PInf6 geneID

C6-C2 PaxiN geneID PInf6 geneID CAD1 Scf00016g02329 Scf01271g08029

PAAS1 Scf00561g00021 Scf00985g02002

CAD2 Scf00152g00245 Scf02279g04026

PAAS2 Scf00152g00237 absent PEP1 Scf00255g01119 Scf02160g01019

PAAS3 Scf00152g00238 Scf01065g00017

PEP2 Scf00089g01842 Scf00281g01022

BPBT1 Scf00007g00012 Scf01180g01014 WRKY3 Scf00073g02335 Scf00191g46015

BPBT2 Scf00007g00011 Scf01180g01013

WRKY4 Scf00732g00236 Scf00782g02035

BPBT3 Scf00169g00922 Scf00276g03003 HSTF-B3 Scf00130g00326 Scf03556g00033

C6-C1

HSF24 Scf00546g00118 Scf00713g03004

PAL1 Scf00123g00096 Scf00871g09016 MYB308 Scf00452g00412 Scf01514g03016

PAL2 Scf00488g00074 Scf00622g01006

IPO1 Scf00170g00623 Scf01154g02016

PAL3 Scf00858g00215 Scf01985g02016 IPO2 Scf00128g01742 Scf00889g01039

CNL1 Scf00294g00032 Scf00099g03010

Transcription factors

CNL2 Scf00784g00010 Scf00123g10004 LHY Scf00092g01519 Scf00559g14015

CNL3 Scf00294g00411 Scf00099g04016

ODO1 Scf00002g00037 Scf00284g00012

CHD1 Scf00231g00330 Scf00961g08042 EOBI Scf00129g01231 Scf02365g00039

CHD2 Scf01363g00003 Scf05015g00020

EOBIb absent Scf05157g00020

KAT1 Scf00052g00819 Scf00191g27016 EOBII Scf00080g00064 Scf00394g13005

BALDH1 Scf00393g00118 Scf00575g02016

EOBV Scf00362g00831 Scf01468g01015

BALDH2 Scf00017g02715 Scf01380g08045 MYB4 Scf01221g00042 Scf00661gX

BSMT1 Scf00047g01123 Scf00686g02008

PH4 Scf00349g00057 Scf02429g00002

BSMT2 Scf00047g00116 absent Shikimate pathway

BSMT3 Scf00047g01129 absent

DAHPS1 Scf00030g01715 Scf00007g07016

BSMT4 Scf03967g00005 Scf00437g04005 DAHPS2 Scf01060g00145 Scf00791g07004

BSMT5 Scf00423g00020 Scf00437g02026

DAHPS3 Scf00381g00086 Scf00091g21009

BSMT6 Scf00423g00119 Scf00437g02028 DHQSI Scf00328g00523 Scf01063g14031

C6-C3

SD1 Scf01067g00113 Scf02339g01032

C4H1 Scf00390g00225 Scf00951g08008 SD2 Scf00780g00005 Scf01925g00005

C4H2 Scf00556g00035 Scf03806g00033

SK Scf00359g00118 Scf01418g03021

C4H2b absent Scf01430g00027 EPSPS1 Scf00959g00022 Scf00364g12027

4CL1 Scf00207g00334 Scf01633g10028

EPSPS2 Scf00013g03114 Scf02633g02011

4CL2 Scf00314g00086 Scf01099g07012 EPSPS3 absent Scf01031g04025

4CL3 Scf00195g01223 Scf01889g04030

CS Scf00747g00122 Scf00274g00004

HCT1 Scf00553g00538 Scf01142g01039 Arogenate pathway

HCT2 Scf00835g00312 Scf01099g09008

CM1 Scf00166g00931 Scf01969g05044

CCoAOMT1 Scf00016g02023 Scf01271g05017 CM2 Scf00495g00010 Scf00660g01021

CCoAOMT2 Scf00450g00032 Scf00887g03019

PPA-AT Scf00183g01421 Scf00258g04021

CCoAOMT3 Scf00003g02440 Scf00019g01024 ADT1 Scf00114g00001 Scf00392g01004

CCR1 Scf00332g00433 Scf01142g06025

ADT1b absent Scf00392g01021

CCR2 Scf00207g00444 Scf01889g05040 ADT2 Scf00389g00425 Scf05304g00006

CFAT Scf00474g00217 Scf00221g12012

ADT3 Scf00002g00514 Scf00872g12002

IGS1 Scf00889g00229 Scf01349g08001 PPY-AT Scf00027g00165 Scf00011g03011

IGS2 Scf00060g00021 Scf00001g26039

Transporters

EGS Scf00020g01714 Scf00318g05021 ABCG1 Scf01060g00147 Scf00791g07017

pCAT Scf00125g00064 Scf00906g07007

CHAPTER 1

24

Supplemental table 2. Comparison of PaxiN and PinfS6 FVBP proteins. See figure 1 and table 1 for abbreviations. PinfS6 ADT1b and EOBIb have been compared to PinfS6 ADT1a and EOBIa. Amino acid sequences have been aligned with ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Conserved is PAM 250 >250.

Biosynthetic genes

PaxiN PinfS6 changes of which similarity amino acids amino acids conserved av. 98.39

PAAS1 506 506 24 16 95.26 PAAS2 506 absent PAAS3 506 506 30 17 94.07 BPBT1 460 460 9 4 98.04 BPBT2 460 460 7 4 98.48 BPBT3 456 456 9 8 98.03 BSMT1 357 357 29 18 91.88 BSMT2 357 absent BSMT3 336 absent BSMT4 360 360 9 7 97.5 BSMT5 358 358 13 5 96.38 BSMT6 358 356 10 6 97.19 CNL1 570 570 16 8 97.19 CNL2 528 587 111 54 78.86 CNL3 510 570 33 15 93.53 CHD1 724 724 8 6 98.9 CHD2 724 713 8 2 98.88 KAT1 462 462 25 15 94.59 CFAT 454 458 23 10 93.61 EGS 308 308 6 2 98.05 IGS1 323 323 9 4 97.21 IGS2 319 319 17 10 94.67

L-phenylalanine

PaxiN PinfS6 changes of which similarity

precursor genes amino acids amino acids conserved av. 98.91

CM1 324 324 5 3 98.46 DAHPS1 533 533 3 1 99.44 ADT1a 424 424 5 2 98.82 ADT1b absent 424 Transcription PaxiN PinfS6 changes of which similarity factors amino acids amino acids conserved av. 98.71

EOBIa 202 202 1 0 99.5 EOBIb absent 201 EOBII 197 197 1 1 99.49 EOBV 267 273 4 2 98.5 MYB4 258 258 4 2 98.45 ODO1 294 294 7 4 97.62