# Specialized metabolite modifications in Brassicaceae seeds and plants: diversity, functions and related enzymes

Léa Barreda, Céline Brosse, Stéphanie Boutet, François Perreau, Loïc Rajjou, Loïc Lepiniec, Massimiliano Corso

► To cite this version:

Léa Barreda, Céline Brosse, Stéphanie Boutet, François Perreau, Loïc Rajjou, et al. Specialized metabolite modifications in Brassicaceae seeds and plants: diversity, functions and related enzymes. Natural Product Reports, 2024, 41 (5), pp.834-859. 10.1039/D3NP00043E. hal-05272500

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# Specialized metabolite modifications in Brassicaceae seeds and plants: diversity, functions and related enzymes

Received 00th January 20xx,
Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

Léa Barreda, Céline Brosse, Stéphanie Boutet, François Perreau, Loïc Rajjou, Loïc Lepiniec, Massimiliano Corso*

Covering: up to 2023

Specialized metabolite (SM) decorations, such as hydroxylation, methylation, glycosylation and acylation, are functional groups that can be added or removed from SM core structures. This review will summarize available knowledge (up to 2023) on the diversity, nature, activity and role of SM decorations in plants and seeds. We will give a comprehensive overview on the impact of decorations on SM diversity, activities and functions, and on the corresponding enzymes, i.e. hydroxylases, methyltransferases, glycosyltransferases and acyltransferases, in Brassicaceae species. Brassicaceae, including model (Arabidopsis thaliana) and crop (Brassica napus, Camelina sativa) plant species, present a large diversity of plant and seed specialized metabolites, which makes them valuable models to study SM decorations. In this review, particular attention is given to the environmental plasticity of specialized metabolite decorations and relative enzymes. Furthermore, a spotlight is given to decorated SMs and related decoration enzymes in seeds of Brassicaceae species. Seeds constitute a large reservoir of beneficial SMs and are one of the most important dietary sources, providing more than half of the world's intake of dietary proteins, oil and starch. Finally, the seed tissue- and stage- specific expressions of *Arabidopsis thaliana* genes involved in SM decoration are presented and discussed in the context of available literature. Given the major role in plant phytochemistry, biology and ecology, SM decorations constitute a subject of study contributing to the research and development in agroecology, pharmaceutical, cosmetics and food industrial sectors.

## Table of contents

1. Introduction

1.1. Scope of this review

2. Specialized metabolite decorations in Brassicaceae species

2.1. Hydroxylation

2.1.1. Enzymes involved in SM hydroxylation: CYP450s hydroxylases and 2-oxoglutarate-Fe(II) dependent oxygenases

2.1.2. Structural diversity and functions of hydroxylated SMs

2.2. Methylation

2.2.1. Enzymes involved in SM methylation: SAM-dependent methyltransferases

2.1.2. Structural diversity and functions of methylated SMs

2.3. Glycosylation

2.3.1. Enzymes involved in SM glycosylation: glycosyltransferases and glucosidases

2.3.2. Structural diversity and functions of glycosylated SMs

2.4. Acylation

2.4.1. Enzymes involved in SM acylation: BAHD acyltransferases and SCPL acyltransferases

2.4.2. Structural diversity and functions of acylated SMs

3. Specialized metabolite decorations in Brassicaceae seeds

3.1. The plasticity of seed specialized metabolite decorations

3.2. Structural diversity and expression patterns of SM-decoration genes in Arabidopsis seeds

4. Conclusions and perspectives

## 1. Introduction

Plants synthetize a large number of natural organic compounds, referred as primary and specialized metabolites. Primary metabolites are involved in essential physiological and developmental processes, such as photosynthesis, respiration, growth, or reproduction ¹,². Thousands of Specialized Metabolites (SMs), formerly named

Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB),
78000 Versailles, France

* For correspondence: M Corso, massimiliano.corso@inrae.fr

Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/x0xx00000x
secondary metabolites or natural products, are produced in plant and seeds, that constitute a large reservoir and source of diversity for these molecules. SMs are critical for the interactions of plants with their environment, ranging from physiological to developmental adaptation to biotic and abiotic environments, and determine the quality of plant and seed products ¹⁻⁴. SMs usually accumulate at low concentrations, and are characterized by specific distributions according to plant families, genera or species ¹,²,⁵.

Based on their chemical structure, SMs are commonly gathered into three main groups namely terpenoids, nitrogen-containing compounds (including alkaloids and glucosinolates) and phenylpropanoids (or phenolic compounds). (Figure 1a) ¹,³. Besides these metabolic classes, plant hormones (e.g. abscisic, jasmonic and salicylic acids) and other compounds derived from primary metabolic pathways could be also included within the SM category, as they can be regarded as SMs adapted for interaction with protein receptors ⁶.

In plants, terpenoids are the largest and most structurally diverse group of SMs, comprising more than 25 000 representatives. Terpenes are synthetized from precursor units of isopentenyl pyrophosphate (IPP) and its functional isomer dimethylallyl pyrophosphate (DMAPP) ¹,³,⁷ which are produced through mevalonic acid (MVA) and methylerthritol phosphate (MEP) pathways ¹,⁷⁻⁹. Terpenes are chemically classified as simple hydrocarbon molecules and/or lipophilic compounds, while terpenoids correspond to terpenes that have undergo some rearrangements such as oxygenation, carbonylation, dehydrogenation, or hydrogenation. However, these two terminologies are mistakenly interchanged in the literature ¹,⁷,⁸. Terpenoid classification is based on the number and structural organization of the C5 isoprene units, that represents the basic building unit for these metabolites. The terpenoid skeletons consists of an assembly of isoprene units that arise to structural diversity with compounds being simple linear hydrocarbons to carbocyclic terpenoids of several dozens of carbons ¹,⁷,⁸. Hemiterpenoids (C5) are the smallest class of terpenoids since they contain solely 1 isoprene unit. Then there are the monoterpenoids (C10) (e.g. limonene), sesquiterpenoids (C15) (e.g. farnesene), diterpenoids (C20) (e.g. geranyllinalool and gibberelins), sesterterpenoids (C25) (e.g. ophiobolin A), triterpenoids (C30) (e.g. squalene and thalianol), tetraterpenoids (C40) (e.g. carotenoids), and finally the polyterpenoids (>C40) (e.g. natural rubber) with more than 8 isoprene molecules (Figure 1a) ¹,⁷,⁸,¹⁰.

Nitrogen-containing compounds constitute the second group of SMs, and includes alkaloids, glucosinolates, cyanogenic glycosides, some non-protein amino acids and some bioactive peptides. Most of the nitrogen-containing compounds are synthetized from amino acids ¹. Among them, alkaloids are the most abundant category with about 20 000 annotated compounds ³,¹¹. Alkaloids, such as caffeine, nicotine or morphine, are heterocyclic compounds, mostly derived from lysine, tryptophan or tyrosine amino acids, containing at least one nitrogen atom in their structure (Figure 1a) ¹. Nevertheless, some alkaloids present skeletons derived from the MVA pathway, mainly associated to the biosynthesis of terpenoids ¹. Glucosinolates

(GSLs) are sulphur-containing nitrogen compounds that are mostly found in Brassicales (including families such as Brassicaceae, Capparaceae or Cleomaceae) species ¹,¹²,¹³. More than 130 structures have been identified so far ¹,¹⁴. GLS are characterized by a β-thioglucoside N-hydroxysulfate core structure, comprising a β-D glucopyranosyl moiety and a side-chain (or “R-group”) that can be aromatic, indolic, or aliphatic derivatives of amino acids precursors (Figure 1a). Methionine, tryptophan and phenylalanine are the main amino acids precursors of GLS, although alanine, valine, isoleucine, leucine, tyrosine and tryptophan are also found (Figure 2) ¹,¹⁴,¹⁵. Another specifity of the GSL is that upon cellular disruption (occurring for instance in an herbivory stress context), they are hydrolysed into isothiocyanates (ITCs), thiocyanates and nitriles by endogenous myrosinase (Figure 2) ¹,¹⁶. Finally, cyanogenic glycosides contain a cyanohydrin group ($R_2C(OH)CN$) in which R correspond to H, alkyl or aryl) in the aglycone (Figure 1a) ¹.

Last, phenolic compounds (or phenylpropanoids) are composed of one or more phenolic groups, corresponding to C6-aromatic rings with one or several hydroxyl groups, that are synthetized through shikimate and malonate pathways ¹,³,¹⁷. It is estimated that the plant kingdom produces over than 10 000 phenolic compounds that are heterogeneously distributed among species ¹,³,¹⁷,¹⁸. The structures of these compounds are diverse, ranging from simple molecules containing one single aromatic ring, such as phenolic acids or cinnamic acids, to complex polymers of basic/simple phenolic molecules forming compounds such as tannins (or proanthocyanidins) (Figure 1a) ¹,³,¹⁹. Flavonoids are the largest phenylpropanoid category, with over 9 000 representatives ³,²⁰,²¹. They have a C6-C3-C6 backbone characterized by two benzene rings (ring A and B) connected by a heterocyclic pyran ring (ring C). Flavonoids can be classified into six main families according to the oxidation level of the pyran ring (C): flavonols, anthocyanidins, isoflavones, flavones, flavanones and flavanols ³,²². Divergences in hydroxylation and methylation patterns of benzene rings (A and C) determine individual compounds within each family (Figure 3) ²⁰,²². Beside flavonoids, other phenolic compounds display a simpler and smaller structure. This is the case of phenolic acids such as benzoic acid and cinnamic acids, presenting respectively C6-C1 and C6-C3 skeletons which correspond to an aromatic/phenyl group fused to a linear side chain (Figure 4)¹,³. They can be differentiated on the basis of their carboxylic group containing chain length. Coumarins, also have C6-C3 type skeleton but are cyclic esters comprising a benzene and a pyran ring and can be classed according to the complexity of their carbon backbone ¹,³. Finally, tannins are phenolic compounds resulting from the association of phenol derived molecules. Condensed tannins (also termed as proanthocyanidins) are polymers of the flavonoid unit flavan-3-ols while hydrolysable tannins consist of glucose esterified to gallic acid (a phenolic acid) ¹,³,¹⁸,¹⁹.

## 1.1 Scope of this review
This review focuses on diversity, nature, activity and role of SM
decorations in plants and seeds. We mainly focus on the addition or
removal of functional groups, which we refer as “decorations” or
“modifications”, to the metabolites. We present an overview of the
enzymes involved in SM decoration reactions. Particular attention is
paid on the functions that can be conferred by the modifications.
<figure><img src="image_2.png" /><figcaption>**Figure 1:** Diversity and structures of specialized metabolites found in Brassicaceae seeds. a) Categories and structures of specialized metabolites found in Brassicaceae. b) Seeds from Brassicaceae species.</figcaption></figure>

We decide to focus on Brassicaceae species. This genus include
*Arabidopsis thaliana*, the model organism for research in plant genetics and molecular biology, that is characterized by a small genome (~125 Mb) and for which a large amount of multiomic data (including genomics, transcriptomics, targeted and untargeted metabolomics) and a large collection of mutants are available and constitute a great resource to characterize plant and seed metabolic pathways<sup>23-27</sup>. Besides the model species *A. thaliana*, many crops belong to the Brassicaceae genus, including many *Brassica* (e.g. *Brassica napus* [rapeseed], *Brassica rapa* and *Brassica oleracea*) and *Camelina sativa* plant species (Figure 1b). Brassicaceae model and crop plants and seeds are rich in oil (lipids and fatty acids) and proteins and present a large diversity of specialized metabolites. Moreover, decorated SM are well represented for all the specialized metabolic categories identified in Brassicaceae species.

In the last part of the review, a spotlight is put on decorated SMs and related decoration enzymes in plant seeds. Seeds contain a vast and diverse number of SMs that are important for their agronomic, physiological, nutritional and/or industrial qualities <sup>3,18,19</sup>. Furthermore, seeds constitute (directly or indirectly) a major source of proteins, oil and starch for both human and animal nutrition. Seeds are the main vectors for improvement of agronomical practices and the management of genetic resources, two major factors for the development of sustainable agriculture and the preservation of biodiversity <sup>28</sup>. Finally, seed storage compounds represent nowadays a sustainable alternative to fossil carbon for chemical industry. For instance, Brassicaceae species are widely cultivated for oil production purpose, leading to interesting recoverable co-products. For all these reasons, Brassicaceae seeds constitute attractive models for both studying and producing wide amounts of interesting SMs.

## 2. Specialized metabolite decorations in Brassicaceae species

Specialized metabolites belonging to each category (e.g. terpenoids, alkaloids, glucosinolates, flavonoids etc.) present a low number of core structures (Figure 1a). However, the large structural diversity observed among SMs arises from various decorations added to the core structures <sup>17,20,29</sup>. Among these, hydroxylations, methylations, glycosylations and acylations are the most frequent (decorations) <sup>4,18,29,30</sup>. The addition of these decorations to SM core structures are usually catalysed by enzymes (e.g. hydroxylases, methyltransferases, glycosyltransferases and acyltransferases). Is it important to mention that spontaneous chemical reactions, notably due to the presence of oxidants, can also lead to the onset of decorations although data about those events are rather scarce <sup>4</sup>. Besides their contribution to the large SMs chemical diversity, metabolite decorations can affect their chemical properties, transport and storage, thus determining their biological activities <sup>4,17,18,20,29</sup>. Given that decorations can determine SMs roles during plant life cycle but also their industrial use (e.g. agricultural, food/feed, cosmetic, and pharmaceutical sectors), studying these decorations is of major importance <sup>22,31,32</sup>.

### 2.1. Hydroxylation

Hydroxylation corresponds to the introduction of a hydroxyl group (-OH) into a molecule through the substitution of functional groups or hydrogen atoms.

#### 2.1.1. Enzymes involved in SM hydroxylation: CYP450s hydroxylases and 2-oxoglutarate-Fe(II) dependent oxygenases

The enzyme families involved in SM hydroxylation include the cytochrome P450 (CYP450)-family enzymes, the 2-oxoglutarate-Fe(II)-ascorbate dependent dioxygenases (2-ODDs) and some monooxygenases such as the flavin-dependent monooxygenases, tyrosinases and peroxidases <sup>20,33,34</sup>. Those enzymes are involved in the hydroxylation of nearly all organic molecules ranging from precursors synthesis to the decoration of SM core structures. Is it worth noting that methylation, glycosylation and acylation usually occur on hydroxyl groups, which explains why hydroxylations usually take place earlier than other modifications in SM pathways <sup>35</sup>. This review gives an overview on the hydroxylases that are well-characterized or putatively involved in SMs hydroxylation decorations.

#### CYP450 hydroxylases

CYP450s constitute an ancient superfamily of heme-thiolate oxidoreductase/monooxygenase enzymes, gathering more than 300 000 sequences <sup>36</sup>, that are present in all life kingdom <sup>37,38</sup>. These proteins were first discovered in animals and named P-450 for light absorption band at 450 nm <sup>38-40</sup>. Further analyses later revealed that CYP450s were not pigments but enzymes involved in a wide range of catalytic reaction pathways <sup>41</sup>. Indeed, CYP450s catalyse over 20 types of reactions including notably hydroxylation, oxygenation, dealkylation and epoxidation <sup>42-45</sup>. Nevertheless, hydroxylation is the main and most frequent reaction catalysed by the P450s, being the largest enzyme family involved in NADPH-and/or O2-dependent hydroxylation reactions across all the domains of life <sup>38,44</sup>. These reactions participate in biosynthetic pathways of lignins, flavonoids, terpenoids, glucosinolates, lipids, and hormones <sup>38,46</sup>. Interestingly, plant CYP450 family is divided into two groups based on evolutionary relationships: Type A and non-A-type <sup>46,47</sup>. Plant-specific A type CYP450s emerged after the divergence of plants from animals and fungi, while non-A type CYP450s display sequence similarities with enzymes found in animals and fungi. In Brassicaceae species, near to 1% of their genomes encode for CYP450 genes (e.g. 0.916 % in *A. thaliana*; 0.8598 % in *B. rapa* or 0,7496 % in *B. oleracea*), highlighting the abundance and suggesting the various functions of CYP450 in plants <sup>35,44,48,49</sup>.
CYP450s are hierarchically classified into clans, families and

<figure><img src="image_3.png" /></figure>
**Figure 2:** Glucosinolate biosynthesis pathway in Arabidopsis. (Adapted from Harun et al., 2019<sup>238</sup>; Chhajed et al., 2020<sup>239</sup>; Hooshmand et al., 2021<sup>240</sup>). Enzymes are represented in bold with arrows indicating the catalysed reactions. Hydroxylases (represented in orange): CYP79B2, cytochrome P450 79B2; CYP79B3, cytochrome P450 79B3; CYP79C1, cytochrome P450 79C1; CYP79C2, cytochrome P450 79C2; CYP83B1, cytochrome P450 83B1; CYP83A1, cytochrome P450 83A1 (Involved in core structure formation); GS-OX, flavin-monooxygenase glucosinolate oxygenase; AOP2, alkenyl hydroxalkyl-producing 2; AOP3, alkenyl hydroxalkyl-producing 3; CYP81F4, cytochrome P450 81F4; CYP81F1, cytochrome P450 81F1; CYP81F2, cytochrome P450 81F2; CYP81F3, cytochrome P450 81F3 (Involved in side chain modifications). Methyltransferases (represented in green): IGMT1, indole GLS O-methyltransferase 1; IGMT2, indole GLS O-methyltransferase 2; IGMT5, indole GLS O-methyltransferase 5; COMT, caffeate/5-hydroxyferulate 3-O-methyltransferase (Involved in side chain modifications). Glycosyltransferases (represented in purple): UGT74B1, UDP-glucosyl transferase 74B1; UGT74C1, UDP-glucosyl transferase 74C1 (Involved in core structure formation). Acyltransferases (represented in blue): SCPL17, serine carboxypeptidase-like 17; BZO1, benzoyloxy GLS 1 (Involved in side chain modifications). Other enzymes (represented in black): BCAT4, branched-chain amino acid aminotransferase 4; MAM, methylthioalkylmalate synthase; IPMDH, isopropylmalate dehydrogenase; IMPI, isopropylmalate hydrogenase, BCAT3, branched-chain amino acid aminotransferase 3 (Involved in side chain elongation); GSTF, glutathione S-transferase F; GGP1, γ-glutamyl peptidase 1; SUR1, super root 1; C-S-lyase; SOT, sulfotransferase (Involved in core structure formation); TGGs, β-thioglucoside glucohydrolases; PEN2/3, penetration 2/3; BGLU, β-glucosidase. AAO4, aldehyde oxidase 4; CHY1, cinnamoyl-CoA hydrolase; 4CL, 4-coumaric acid: CoA ligase (Involved in side chain modification). Abbreviation of glucosinolate (GLS) names: 1OH1M 1-hydroxyindol-3-ylmethyl GLS; 4OH1M 4-hydroxyindol-3-ylmethyl GLS; 1MO1M 1-methoxyindole-3-ylmethyl GLS; 4MO1M 4-methoxyindole-3-ylmethyl GLS; 3MTP, 3-methylthiopropyl GLS (Glucoiberverin); 4MTB, 4-methylthiobutyl GLS (Glucoerucin); 5MTP, 5-methylthiopentyl GLS (Glucoberteroin); 6MTH, 6-methylthiohexyl GLS (Glucolesquerellin); 7MTH, 7-methylthioheptyl GLS; 8MTO, 8-methylthiooctyl GLS; 3MSOP, 3-methylsulfinylpropyl GLS (Glucoiberin); 4MSOB, 4-methylsulfinylbutyl GLS (Glucoraphanin); 5MSOP, 5-methylsulfinylpentyl GLS (Glucoalyssin); 6MSOH, 6-methylsulfinylhexyl GLS (Glucohesperin); 7MSOH, 7-methylsulfinylheptyl GLS (Glucoibarin); 8MSOO, 8-methylsulfinyloctyl GLS (Glucohirsutin); 2PROP, 2-propenyl GLS (Sinigrin); 3OHP, 3-hydroxypropyl GLS; 4OHB, 4-hydroxybutyl GLS; 3UT, 3-butenyl GLS (Gluconapin); 2-OH-3BUT, (2R)-2-hydroxy 3-butenyl GLS (Progoitrin); 4-PENT, 4-pentenyl GLS (Glucobrassicanapin); 2OH-4PENT (Gluconapoleiferin); 3BZO, 3-benzoyloxypropyl GLS (Glucomalcomiin); 3SIN, 3-sinapoyloxypropyl GLS; 4BZO, 4-benzoyloxybutyl GLS; 4SIN, 4-sinapoyloxybutyl GLS. BzH, benzaldehyde; BzOH, benzoic acid; BzCoA, benzoyl-coenzyme A.

subfamilies, which are reported in their “CYP” code. Enzymes sharing 40% of amino acid similarity are considered members of the same family. In plants, CYP450 are attributed to families with the numbers 51, 71-99, 701-999 and from 7001 onward. Enzymes sharing >55% belong to the same subfamily, which is defined by a letter (e.g. CYP709A belong to the subfamily A from 709 family). Moreover, newly discovered CYP450 genes sharing >97% similarity with another previously identified CYP450 gene are considered as allelic variants<sup>35,38,44</sup>. Finally, CYP450 clans are defined by the deepest distinct branching phylogenetic clades that are reproducible in various phylogenetic trees (Supplementary Figure S1)<sup>35</sup>. In Brassicaceae, CYP450 enzymes are divided into 9 clans. Among them, CYP71, CYP72, CYP85 and CYP86 are multifamily members and their names correspond to their lowest-numbered family members. The other six clans are named based on their family number such as CYP51, CYP74, CYP97, CYP710 and CYP711. Clans 71, 72, and 85 constitute the largest CYP clans in *A. thaliana*, *B. rapa* and *B. oleracea*<sup>49</sup>. Moreover, these clans include several taxa-specific (sub)families resulting from lineage-specific evolutions. In this way, clan 71 is considered as specific to plants and includes families that are restricted to land plants such as CYP73 and CYP78. Moreover, (sub)families such as CYP702, CYP708 and CYP705 are Brassicaceae specific<sup>35</sup>. Inversely, some CYP families and/or clans can be common to larger classification of organisms. CYP51 clan is, for instance, ubiquitous among kingdoms<sup>35</sup>.

Substrates and activities of a wide range of plant CYP450s have been characterized and reported. Enzymes belonging to the same family usually shared similar both enzymatic activities and substrates. CYP450s characterized in Brassicaceae and corresponding references are detailed in Supplementary Table S1. Several clan 71 enzymes from families CYP79, CYP81 and CYP83 and families CYP73, CYP75, CYP82, CYP84 and CYP98 are involved in glucosinolate (SMs specific to Brassicaceae ssp) and phenylpropanoid hydroxylations, respectively (Figures 2, 3 and 4)<sup>50-53</sup>. CYP71 family, which is the largest family with 50 members in *A. thaliana*, include enzymes involved in alkaloid and terpenoid hydroxylations. Other families,

namely CYP72, CYP76, CYP82, CYP88, CYP96, CYP97, CYP701, CYP705, CYP706, CYP708, CYP714 and CYP716 are able to hydroxylate terpenoid compounds<sup>46,54</sup>. Families CYP51, CYP85, CYP90, CYP710 and CYP734 are involved in steroid hydroxylations<sup>35,46</sup>. Some CYP450s from clans 71 (families CYP77, CYP78, CYP703 and CYP704), 74 (family CYP74) and 86 (families CYP86 and CYP704) are involved in the hydroxylation of fatty acids during their biosynthesis<sup>35,46</sup>. Finally, some enzymes are involved in the biosynthesis of hormones. For example, CYP707, CYP94 and CYP711 families are involved in the biosynthesis of abscisic acid, jasmonate and strigolactones, respectively<sup>35,46</sup>.

## 2-oxoglutarate-Fe(II) dependent oxygenases

2-oxoglutarate/Fe(III)-dependent dioxygenases (2-ODDs) constitute a large superfamily of non-heme proteins that, similarly to the CYP450, are present in all life kingdoms. In plants, they are encoded by the second largest family of genes, following CYP450s superfamily<sup>55</sup>. 2-ODDs catalyse a wide range of oxidoreduction reactions including hydroxylation, dealkylation, desaturation, epoxidation, epimerization, halogenation, cyclization, peroxide formation and ring expansion/contraction<sup>56,57</sup>. However, as for CYP450s, hydroxylation remains the predominant reaction performed by this class of enzymes. Distinctly from CYP450s, which are monooxygenases, 2-ODDs are dioxygenases and have therefore the ability to incorporate two oxygen atoms into their corresponding substrates. Moreover, as suggested by their name, 2-ODDs use 2-oxoglutarate and dioxygen as substrates, and Fe(II) as co-factor, yielding succinate and carbon dioxide as co-products<sup>56,58</sup>. Considering both their substrate and product specificities, 2-ODDs are highly “biochemically flexible” enzymes. They can indeed take part to a wide range of reactions and modify the biological function (by hydroxylation) of a large number of specialized metabolites. Besides their role in nucleic acid repair, epigenetic regulation, transcriptional regulation and protein biosynthesis, they are involved in several specialized metabolite (and lipid) biosynthetic and detoxification pathways<sup>56,58,59</sup>. Although 2-ODD amino acid
sequences are highly divergent, they present a conserved trial motif including 2 His and one Asp or Glu residue (HXD/EXnH), housed in a double-stranded β-helix (DSBH) fold, which is involved in Fe(II) chelation<sup>57,60,61</sup>. A detailed phylogenetic classification of hundreds of

2-ODDs from six model plants, ranging from green algae to angiosperms, has been performed<sup>61</sup>. 2-ODDs were classified into three main classes: DOXA, DOXB and DOXC. Enzymes of the DOXA class are suggested to be involved in N-demethylating DNA repair,

<figure><img src="image_4.png" /><figcaption>**Figure 3:** Flavonoid biosynthesis pathway in Arabidopsis. (Adapted from Lepiniec et al., 2006¹⁹; Tohge et al., 2017²¹; Saigo et al., 2020²⁴¹). Enzymes are represented in bold with arrows indicating the catalysed reactions. Hydroxylases (represented in orange): F3H, flavanone 3-hydroxylase; F3'H, flavonoid 3'-hydroxylase (CYP75B1); FLS, flavonol synthase; LDOX, leucoanthocyanidin dioxygenase; ANS, anthocyanidin synthase; C3'H, coumarate 3' hydroxylase (CYP98A3); C4H, cinnamic acid 4-hydroxylase (CYP73A5); F6'H, feruloyl-CoA 6'-hydroxylase; S8H, scopoletin 8-hydroxylase. Methyltransferases (represented in green): OMT1, O-methyltransferase 1; CCoAOMT7, caffeoyl-CoA O-methyltransferase 7; CCoAOMT, caffeoyl CoA 3-O-methyltransferase. Glycosyltransferases (represented in purple): F7RhaT, Flavonol 7-O-rhamnosyltransferase (UGT89C1); F7GlcT, Flavonol 7-O-glucosyltransferase (UGT73C6); F3RhaT, Flavonol 3-O-rhamnosyltransferase (UGT78D1); BGLU6, Flavonol 3-O-glucoside:6''-O-glucosyltransferase; F7GlcT, Flavonol 7-O-glucosyltransferase (UGT73C6); F7RhaT, Flavonol 7-O-rhamnosyltransferase (UGT89C1); F3AraT, Flavonol 3-O-arabinosyltransferase (UGT78D3); A3GlcT, Anthocyanidin 3-O-glucosyltransferase (UGT78D2); A3GlcRhaT Anthocyanin 3-O-glucoside: 3''-O-rhamnosyltransferase (UGT79B2, UGT79B3); A3Glc:2''XylT, Anthocyanin 3-O-glucoside:2''-O-xylosyltransferase (UGT79B1); A3RhaT, Anthocyanin 3-O-rhamnosyltransferase (UGT79B2, UGT79B3); A5GlcT, Anthocyanin 5-O-glucosyltransferase (UGT75C1); BLGU10, Anthocyanin 3-O-(p-coumaroyl) glucoside:glucosyltransferase. Acyltransferases (represented in blue): HCT, hydroxycinnamoyl-Coenzyme A shikimate/quinate hydroxycinnamoyltransferase; S8H, scopoletin hydroxylase; FPT2, flavonol-phenylacetyltransferase 2 A3Glc:6''AcylT, hydroxycinnamoyl-CoA:anthocyanin 3-O-glucoside:6''-O-acyltransferase; A5Glc:6''MalT, Anthocyanin 5-O-glucoside:malonyltransferase. Other enzymes (represented in black): PAL, phenylalanine ammonia-lyase; 4CL, 4-coumaric acid: CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; LAR, leucoanthocyanidin reductase. Legend for flavonoid structure name abbreviations: F, Flavonol; A, Anthocyanidin; T, transferase; Glc, Glucose; Rha, Rhamnose; Ara, Arabinose; Xyl, Xylose; pCou, pCoumaroyl; Caf, Caffeoyl; Sin, Sinapoyl; Mal, Malonyl</figcaption></figure>
since they belong to the same clade as *Escherichia coli* AlkB, which is known to perform oxidative demethylation of alkylated nucleic acids ⁶¹. The DOXB class is conserved among all plant taxa and was suggested to be involved in cell wall protein modification since it contains prolyl 4-hydroxylase enzymes ⁶¹. Finally, differently from DOXA and DOXB enzymes, which are restricted to basic cell function, DOXC enzymes present various roles and substrates including both primary and specialized metabolites ⁶¹. The enzymes from this 2-ODD category share an N-terminal conserved motif that have been named DIOX_N (Pfam id : PF14226) ⁶¹. Higher phylogenetic resolution of DOXC enzymes allowed the identification of over 50 sub-clades of characterized or putative 2-ODDs. Interestingly, DOXC distributions was found to be highly variable from clades being present throughout land plants, such as DOXC21, DOXC23 and DOXC45, to single species occurrence ⁶¹. Clades DOXC16 and DOXC24, both containing 2 uncharacterized genes, seem to be specific to A. thaliana ⁶¹. From the 130 2-ODD genes, representing around 0.5% of the genome complement, 96 belong to DOXC category ⁶¹. More than 30 DOXC A. thaliana enzymes have already been characterized to date (Supplementary Table S1). DOXC3, DOXC7 and DOXC12 clades include enzymes that are involved in gibberellin biosynthesis, whereas DOXC28, DOXC30 and DOXC47 contain enzymes participating in flavonoid biosynthesis pathways ⁶¹. Moreover, enzymes involved in glucosinolate metabolism are found in both DOXC31 and DOXC20 clades. Finally, DOXC38 and DOXC53 clades contain enzymes involved respectively in ethylene and salicylic acid hormones biosynthesis (Supplementary Table S1) ⁶¹. Phylogenetic tree of A. thaliana 2-ODDs is presented in Supplementary Figure S1.

### 2.1.2. Structural diversity and functions of hydroxylated SMs

Hydroxylation alters SM physico-chemical properties, hence affecting the activities and biological functions of such metabolites (Table 1) ⁴. Hydroxylation is undoubtedly the most common modification of flavonoids. Interestingly, the proportion of flavonoid metabolites that are hydroxylated (and glycosylated) are quite constant across plant species, whereas methylation and acylation are less common and tend to be restricted to fewer plant species ²⁰. Hydroxylation of flavonoids increase their hydrophilic potential ⁴,¹⁸. For instance, quercetin hydroxylation increases water solubility by a factor of 1.5 to 2, depending on the position of the hydroxyl group ¹⁸. Moreover, hydroxylation could increase the antioxidant capacities of some flavonoids ⁶². Quercetin (3',4'-OH) scavenging activity is for instance stronger than kaempferol scavenging activity (4'-OH) ⁶³,⁶⁴. Interestingly, the antioxidant capacities can differ depending on hydroxyl group positions within flavonoids aromatic rings. Hydroxylation occurring in both A and B rings induced a higher increase of the antioxidant capacity compared to the hydroxylation occurring in C ring ¹⁸,²⁰. Free radical quenching capacities of flavonoids positively correlate with the presence of hydroxyl groups in A and B rings ⁶⁵. Hence, the effects of hydroxylation on SM functions are tightly related to its contribution to their antioxidant capacities. Moreover, SM hydroxylation can affect plant protection against biotic stress. Hydroxylation can increase antipathogenic and

antifungal properties of the concerned SM. For instance, the hydroxylated form of the indole GLS indol-3-ylmethyl, 1-hydroxyindol-3-ylmethyl GLS (1OH13M) which is produced by CYP81F4, have toxic and/or deterrent effects against nematodes in A. thaliana (Figure 2) ⁶⁶. However, is it worth to note than the authors also discovered 1OH13M derivatives (including glycosylated derivatives) that could, at least in part, contribute to the antipathogenic properties observed ⁶⁶.

While in some cases hydroxylation activates SMs, there are also some examples where the addition of a hydroxyl group reduced SM activity against stress tolerance. This is the case of camalexin, which is among the few indole alkaloids that can be found in Brassicaceae species and constitute the major phytoalexin of A. thaliana ⁶⁷. Camalexin production is induced by biotic stresses such as Botrytis cinerea or Alternaria brassicicola fungi infections ⁶⁸⁻⁷⁰. Variation in camalexin biosynthesis correlates with susceptibility to Botrytis in Arabidopsis accessions ⁶⁹. Interestingly, it has been proposed that Rhizoctonia solani can hydroxylate and thus detoxify camalexin ⁷¹ Similarly to camalexin, the phytohormone salicylic acid (SA), involved in plant defences against biotrophic pathogens, is inactivated by hydroxylation ⁷². In particular, the hydroxylated SA derivatives 2,5-dihydroxybenzoic acid (2,5-DHBA) and 3-dihydroxybenzoic acid (2,3-DHBA) are respectively produced by SALICYLIC ACID 5-HYDROXYLASE (S5H, also known as DMR6) and SALICYLIC ACID 3-HYDROXYLASE (S3H) ⁷²⁻⁷⁴. SA hydroxylation plays a role in keeping basal SA levels and allow to maintain a balance between growth and immunity ⁷². Interestingly, some bacterial and fungal pathogens such as Ralstonia solanacearum (responsible for tomato wilt disease) possess SA hydroxylases (e.g. NahG) allowing them to inactivate SA ⁷⁵,⁷⁶.

SM hydroxylation can also affect plant responses to abiotic stresses. For instance, it has been suggested that the number of hydroxyl groups on flavonoids correlates with their antioxidant properties and UV radiation tolerance. Indeed, dihydroxy B-ring flavonoids such as quercetin 3-O and luteolin 7-O-glycosides present higher ROS scavenging activities than the monohydroxy B-ring flavonoids such as apigenin 7-O-glycosides and kaempferol 3-O-glycosides ²⁰,⁷⁷. Moreover, under drought and temperature (cold or heat) stresses, ABA 8'-hydroxylase (ABA8OX), belonging to CYP707 family, is known to convert the phytohormone ABA into 8'-hydroxy ABA which is afterwards isomerized to phaseic acid ⁴⁴,⁷⁸,⁷⁹. Besides their antioxidant activity, flavonoids are able to chelate trace metal elements (e.g. Zn and Cu) ⁸⁰⁻⁸³. Metal binding sites have being found to correlate with the number of hydroxyl groups present in a flavonoid structure ²⁰,⁸⁴. Hydroxylation of 3 position of the C-ring is most likely the site showing higher activity in in metal ions chelation ²⁰,⁸⁴. Interestingly, the presence of chelated metal cations such as Fe²⁺, Cu²⁺, Zn²⁺ or Al³⁺ in flavonoid structure allows to block lipid peroxidation which depends on Fe²⁺ and Fe³⁺ ⁸⁵.

Hydroxylation is also the first decoration occurring to carotenoids, turning carotenes into xanthophylls (oxygenated carotenoids). Beta-carotene is hydroxylated at both end-cycles of its carbon chain by CHYB (carotene β-hydroxylase, a non-haeme diiron monooxygenase)
<figure><img src="image_5.png" /><figcaption>**Figure 4:** Cinnamic acid biosynthesis pathway in *Arabidopsis*. (Adapted from Barros et al., 2019<sup>242</sup>; Fraser et al., 2007<sup>183</sup>) Enzymes are represented in bold with arrows indicating the catalysed reactions. Hydroxylases (represented in orange): C4H, cinnamic acid 4-hydroxylase (CYP73A5); C3'H, coumarate 3' hydroxylase (CYP98A3); F5H, ferulic acid 5-hydroxylase (CYP84A1). Methyltransferases (represented in green): COMT, caffeate/5-hydroxyferulate 3-O-methyltransferase; CCoAOMT, caffeoyl CoA 3-O-methyltransferase. Glycosyltransferases (represented in purple): SGT, sinapic acid:UDP-glucose glucosyltransferase (UGT84A2). Acyltransferases (represented in blue): SCT, 1-O-sinapoyl-b-glucose:choline sinapoyltransferase (SNG2, SCPL19); SMT, 1-O-sinapoyl-b-glucose:L-malate sinapoyltransferase (SNG1, SCPL8); SST, sinapoyl-Glc:sinapoyl-Glc sinapoyltransferase (SCPL9); SAT, 1-O-sinapoyl-b-glucose:anthocyanin sinapoyltransferase (SCPL10); HCT, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase; HQT, hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase. Other enzymes: PAL, phenylalanine ammonia-lyase; PTAL, bifunctional phenylalanine/tyrosine ammonia-lyase; 4CL, 4-coumaric acid: CoA ligase; CCR, cinnamoyl CoA reductase; CAD, cinnamyl alcohol dehydrogenase</figcaption></figure>

to produce zeaxanthin, whereas alpha-carotene is hydroxylated by
CHYB and CHYE (carotene c-hydroxylase, which is a cytochrome P450-type monooxygenase), each being specific for one of its two different end-cycles, producing lutein. Zeaxanthin and lutein by different mechanisms can quench chlorophyll triplet produced from light excess, preventing ROS formation and damage, hence the Arabidopsis mutant *npq1.lut2.1* deficient in both xanthophylls show a very low tolerance to light stress⁸⁶.

## 2.2. Methylation

Methylation involves the transfer of a methyl group (-CH3) from a donor to an acceptor molecule. S-adenosyl-L-methionine (SAM or AdoMet) is a major methyl donor due to the high reactivity of its methylated sulfur (sulfonium) moiety towards the target nucleophilic atoms of various molecules⁸⁷⁻⁸⁹.

### 2.2.1. Enzymes involved in SM methylation: SAM-dependent methyltransferases

The enzymes catalysing the transfer of methyl groups using SAM molecules as donors are referred as SAM-dependent methyltransferases (MTs; EC 2.1.1). According to their substrate specificity, MTs can be classified in four groups: N-MTs, O-MTs, S-MTs and C-MTs which incorporate methyl groups into nucleophilic N, O, S or C atoms of the target molecules, respectively ⁸⁸⁻⁹⁰. Moreover, is it worth noting that there are some few MTs that can methylate halogen atoms (Br, Cl, I, etc.), although they are rather scarce in nature ⁹¹,⁹². In *A. thaliana* genome, and when considering other Brassicaceae and all the life domains, O-MTs and N-MTs are the largest MT families ⁸⁸,⁹⁰,⁹¹. Methylation of small organic molecules, such as plant SMs, can be catalysed by all MT enzymes ⁹¹. Nevertheless, O-MTs and N-MTs are the main enzyme families that use SMs as substrate in plants ⁸⁹,⁹³⁻⁹⁶. Hence, we mainly focus on the O-MTs (See Supplementary Figure S1 for *A. thaliana* O-MTs phylogenetic tree) and, to a lesser extent, on the N-MTs. O-MTs are known to add methyl moieties to oxygen atoms from hydroxyl groups of alkaloids, phenylpropanoids (including flavonoids, cinnamic acids or simpler phenols which can notably constitute lignin precursors) phytohormones ⁸⁹,⁹⁵,⁹⁶. Plant O-MTs can be grouped in two major classes. The class I includes mostly enzymes that target hydroxycinnamoyl-CoA esters, namely the CoA O-MTs together with carboxylic acids O-MTs (CO-MTs)⁹⁵⁻⁹⁷. More precisely, CoA O-MTs methylate phenolic hydroxyl groups of hydroxycinnamoyl-CoA esters ⁹⁵. On the other hand, CO-MTs methylate the aliphatic carboxyl groups of benzoic acids, and of salicylic (SA), indoleacetic (IAA) and jasmonic (JA) acids ⁹⁵. The class II is a collection of enzymes acting on a much larger number of metabolite classes, including those involved in phenylpropanoid and alkaloid biosynthesis ⁹⁵⁻⁹⁷. Class I O-MTs are characterized by a lower subunit molecular weight (26-30 kDa), harbour cation dependent activity and do not present the five-consensus sequence motif characteristic of class II O-MTs ⁹⁵⁻⁹⁸. Class II O-MTs molecular weights range from 37 to 43 kDa and present a cation-independent activity ⁹⁶. Class I O-MT enzymes are thought to

have evolved during terrestrial habitat colonization whereas Class II O-MTs have appeared later to face a larger set of evolutionary pressures ⁹⁵⁻⁹⁷. Many common and specific consensus sequence motifs important for SAM binding or catalytic activity, have been characterized for class I and class II O-MTs ⁹⁶. Besides, some O-MTs identified substrate binding sites are also reported ⁹⁶. Interestingly, plant O-MTs generally use a wide range of substrates among alkaloids and phenylpropanoids ⁸⁹,⁹⁶, including Cinnamoyl CoA, Hydroxycinnamic acid and Flavonoid O-MTs (Figures 3 and 4)⁹¹,⁹⁴⁻⁹⁶,⁹⁹. Alkaloid O-MTs have also been characterized ⁹⁷. O-MTs characterized to date in Brassicaceae are presented in Supplementary Table S1. Further information, including information for O-MTs in non-Brassicaceae plant species can be found in recent and detailed reviews from Liu et al. (2022)⁹⁶ for flavonoid O-MTS and Morris and Facchini (2019)⁹⁷ for alkaloid O-MTs.

Although not being as numerous as O-MTs, some N-MTs are crucial for the biosynthesis of alkaloid compounds and derivatives such as caffeine ⁹¹,⁹⁷.

### 2.1.2. Structural diversity and functions of methylated SMs

Methylation modifies the physico-chemical properties of SMs, affecting in turn their bioactivity and bioavailability (Table 1) ²⁰,¹⁰⁰. The less polar methoxy groups (-O-CH₃) commonly replace the highly polar hydroxyl groups, thus impacting SMs hydrophobicity. As an example, the flavonol isorhamnetin, that is an O-methylated quercetin, has slightly higher hydrophobicity (+0.15 for log P) and reduced water solubility (by a 1.7 factor) than quercetin ¹⁸. Methylation constitutes a widespread and efficient method to inactivate and control the homeostasis of several plant hormones such as auxin (indole-3-acetic acid; IAA), gibberellins (GAs) and jasmonates (JAs) ¹⁰¹⁻¹⁰⁵. Nevertheless, methylation does not always have a negative impact on the activity of plant hormones. This is the case of salicylic acid (SA) involved in biotic stress responses, or the strigolactone carlactone involved in shoot branching ⁷²,¹⁰⁶. Indeed, similarly to SA, methyl-salicylate (MeSA) plays an active role in plant defence to cope with biotic stresses ¹⁰⁷⁻¹⁰⁹. Unlike SA, MeSA is a volatile compound released by plants during pathogen infection ¹⁰⁷,¹⁰⁹. In *A. thaliana*, methylation of SA is mediated by BSMT1 (SALICYLIC ACID/BENZOIC ACID CARBOXYL METHYLTRANSFERASE 1). Besides, an esterase named SABP2 (SA-BINDING PROTEIN 2) has been found to reverse SA methylation in tobacco (*Nicotiana tabacum*) suggesting that such reversibility and enzyme could be present in other plants ¹¹⁰. Moreover, SM MTs genes and/or the corresponding methylated-SM have been described to be involved in abiotic and biotic stress responses in Brassicaceae. For instance, the caffeoyl CoA O-MT CCoAOMT1, responsible for the formation of feruloyl CoA from caffeoyl CoA, is known to play a crucial role in *A. thaliana* tolerance to high salinity stress (Figure 4) ¹¹¹. CCoAOMT1 gene expression was up-regulated by saline stress in *A. thaliana* root cell suspension and *A. thaliana* *coaomt1* KO mutant showed hypersensitivity to salt stress ¹¹¹. More recently, a role for CCoAOMT1 during drought stress was suggested in *A. thaliana* ¹¹². As
in the case of high salinity stress, CCoAOMT1 transcription is induced by drought stress and *coaomt1* mutants display hypersensitivity to water deprivation. The authors suggested that CCoAMT1 has a positive role in response to drought stress by regulating H₂O₂

accumulation and acid abscisic (ABA) signalling ¹¹². Besides caffeoyl CoA derivatives, the methylated/methoxylated indole GLS 4-methoxyindole-3-ylmethyl glucosinolate (4MOI3M) is known to be involved in biotic stress response ¹¹³,¹¹⁴. Indeed, the myrosinase

<table><caption>**Table 1:** Effects of decorations on specialized metabolites. Examples of decorated specialized metabolites are listed. The conferred function/effect observed upon decoration is detailed. Enzymes involved in corresponding decorations are indicated if known.

(Continued on next page)</caption><thead><tr><th rowspan="2">Decoration category</th><th rowspan="2">Decorated Category</th><th colspan="6">Functions / Type</th><th rowspan="2">Description / details</th><th rowspan="2">Associated enzyme(s)</th><th rowspan="2">References</th></tr><tr><th>Activation</th><th>Inactivation</th><th>Biotic stress</th><th>Abiotic stress</th><th>Transport</th><th>Solubility</th><th>Volatility</th><th>Stability</th><th>Antioxidant</th><th>Chelation</th></tr></thead><tbody><tr><td rowspan="13">Glycosylation</td><td>Flavonoids (flavonones, flavonols, anthocyanins)</td><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>Transport to vacuoles</td><td>ABCC2 (AT2G34660)</td><td>131</td></tr><tr><td>Flavonoids (flavonols, anthocyanins)</td><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>Transport to vacuoles</td><td>TT12 (AT3G59030)</td><td>130</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Salt stress tolerance and oxidative stress response</td><td>UGT71B1 (AT3G21750)</td><td>121</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Oxidative stress response</td><td>UGT71B2 (AT3G21760)</td><td>121</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Oxidative stress response</td><td>UGT71B7 (AT3G21790)</td><td>121</td></tr><tr><td>Cinnamic acid derivatives (Caffeic acid), Flavonoids</td><td></td><td></td><td>x</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td>Salt stress tolerance</td><td>UGT71C1 (AT2G29750)</td><td>121, 153</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td>Salt stress tolerance</td><td>UGT71C2 (AT2G29740)</td><td>121</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td>Salt stress tolerance</td><td>UGT71C3 (AT1G07260)</td><td>121</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td>Salt stress tolerance</td><td>UGT71D1 (AT2G29730)</td><td>121</td></tr><tr><td>Xenobiotics (3,4-dichloroaniline)</td><td></td><td></td><td>x</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td>Xenobiotic detoxification</td><td>UGT72B1 (AT4G01070)</td><td>163</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td>Salt stress tolerance</td><td>UGT72B2 (AT1G01390)</td><td>121</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td>Oxidative stress response</td><td>UGT73B2 (AT4G34135)</td><td>121</td></tr><tr><td>Flavonoids (Quercetin)</td><td></td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>Drought stress tolerance</td><td>UGT73B3 (AT4G34131)</td><td>121, 138</td></tr><tr><td>Xenobiotics (2,4,6-trinitrotoluene)</td><td></td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>Xenobiotic detoxification</td><td>UGT73B4 (AT2G15490)</td><td>121, 162</td></tr><tr><td></td><td></td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>Hypersensitive response, Drought stress tolerance, Oxidative stress response</td><td>UGT73B5 (AT2G15480)</td><td>121, 138, 149</td></tr><tr><td></td><td>Xenobiotics (2-hydroxylaminodinitrotoluene, 4-hydroxylaminodinitrotoluene, 2-aminodinitrotoluene, 4-aminodinitrotoluene)</td><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>Xenobiotic detoxification</td><td>UGT73C1 (AT2G36750)</td><td>121, 162</td></tr><tr><td></td><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Salt stress tolerance, Oxidative stress response</td><td>UGT73D1 (AT3G53150)</td><td>121</td></tr><tr><td></td><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Salt stress tolerance, Oxidative stress response</td><td>UGT74D1 (AT2G31750)</td><td>121</td></tr><tr><td></td><td>Hormones (Indole-3-butyric acid)</td><td></td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>Drought stress tolerance</td><td>UGT74E2 (AT1G05680)</td><td>121, 145</td></tr><tr><td></td><td>Hormones (Salicylic acid, Anthranilate)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Salt stress tolerance</td><td>UGT74F1 (AT2G43840)</td><td>141</td></tr><tr><td></td><td>Hormones (Salicylic acid, Anthranilate)</td><td></td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>Salt stress tolerance</td><td>UGT74F2; SGT1 (AT2G43820)</td><td>121, 141, 146</td></tr><tr><td></td><td>Flavonoids (Quercetin glycosides)</td><td></td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>UV stress tolerance</td><td>UGT75B1 (AT5G07990)</td><td>158</td></tr><tr><td></td><td>Hormones (Indole-3-butyric acid)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Salt stress tolerance</td><td>UGT75D1 (AT4G15550)</td><td>121, 147</td></tr><tr><td></td><td>Hormones (Isoleucic acid (2-hydroxy-3-methyl-pentanoic acid))</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Salt stress tolerance</td><td>UGT76B1 (AT3G11340)</td><td>139, 140</td></tr><tr><td></td><td>Hormones (Jasmonic acid)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Oxidative stress response</td><td>UGT76E1 (AT5G59580)</td><td>121</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Salt stress response</td><td>UGT76E9 (AT5G38010)</td><td>121</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Oxidative stress response</td><td>UGT76F2 (AT3G55700)</td><td>121</td></tr><tr><td></td><td>Flavonoids (Quercetin, Kaempferol)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Oxidative stress response</td><td>UGT78D1; AtF3RhaT (AT1G30530)</td><td>121, 151</td></tr><tr><td></td><td>Flavonoids (Flavonols)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Cold stress tolerance</td><td>UGT78D3; AtF3AraT (AT5G17030)</td><td>121, 126</td></tr><tr><td></td><td>Flavonoids (Anthocyanidins : Cyanidin 3-O-glucoside)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Drought stress tolerance</td><td>UGT79B1; AtA3Glc:2"XylT (AT5G54060)</td><td>121, 153</td></tr><tr><td></td><td>Flavonoids (Anthocyanidins : Cyanidin, Cyanidin 3-O-glucoside)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Thermic (heat and cold) stress tolerance</td><td>UGT79B2 (AT4G27560)</td><td>121, 160</td></tr><tr><td></td><td>Flavonoids (Anthocyanidins : Cyanidin, Cyanidin 3-O-glucoside)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Stabilization of anthocyanidins - Enhancing of antioxidant capacities</td><td>UGT79B3 (AT4G27570)</td><td>160</td></tr><tr><td></td><td>Cinnamic acid derivatives (Cinnamic acid, p-coumaric acid, Caffeic acid, Ferulic acid, Sinapic acid)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Heat stress tolerance</td><td>UGT81A1 (AT4G31780)</td><td>121</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>UV stress tolerance</td><td>UGT84A1 (AT4G15480)</td><td>121, 150, 152</td></tr><tr><td></td><td>Cinnamic acid derivatives (Sinapic acid)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>UV stress tolerance, Salt stress tolerance</td><td>UGT84A2 (AT3G21560)</td><td>121, 150, 152</td></tr><tr><td></td><td>Cinnamic acid derivatives (Cinnamic acid, p-coumaric acid, Caffeic acid, Ferulic acid, Sinapic acid)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>UV stress tolerance, Cold stress tolerance</td><td>UGT84A3 (AT4G15490)</td><td>121, 150, 152</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>UV stress tolerance, Salt stress tolerance</td><td>UGT84A4 (AT4G15500)</td><td>121, 152</td></tr><tr><td></td><td>Hormones (Cytokinins)</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Heat stress tolerance</td><td>UGT85A1 (AT1G22400)</td><td>121, 161</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Heat stress tolerance</td><td>UGT85A2 (AT2G30140)</td><td>121</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Salt stress tolerance (in tobacco)</td><td>UGT85A5 (AT1G22370)</td><td>148</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Heat stress tolerance</td><td>UGT86A2 (AT2G28080)</td><td>121</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Oxidative stress response</td><td>UGT87A1 (AT2G30150)</td><td>121</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Oxidative stress response</td><td>UGT88A1 (AT3G16520)</td><td>121</td></tr><tr><td></td><td>Flavonoids (Flavonols)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>UGT89C1; AtF7RhaT (AT1G06000)</td><td>121</td></tr><tr><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Thermic (heat and cold) stress tolerance</td><td>UGT90A1 (AT2G16890)</td><td>121</td></tr><tr><td></td><td>Akaloids (Camalexin)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Unknown</td><td></td><td>69, 71</td></tr></tbody></table>
<table><caption>Table 1: Effects of decorations on specialized metabolites (Continued)</caption><thead><tr><th rowspan="2">Decoration category</th><th rowspan="2">Decorated Category</th><th colspan="6">Functions / Type</th><th rowspan="2">Description / details</th><th rowspan="2">Associated enzyme(s)</th><th rowspan="2">References</th></tr><tr><th>Activation</th><th>Inactivation</th><th>Biotic stress</th><th>Abiotic stress</th><th>Transport</th><th>Stability</th><th>Volatility</th><th>Stability</th><th>Antioxidant</th><th>Chelation</th></tr></thead><tbody><tr><td rowspan="10">Methylation</td><td>Hormones (MeAAs)</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>Hormone homeostasis</td><td></td><td>104</td></tr><tr><td>Hormones (MeGAs)</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>Hormone homeostasis</td><td>AtGAMT1 (AT4G26420), AtGAMT2 (AT5G56300)</td><td>103</td></tr><tr><td>Hormones (MeCarlolactone)</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>Hormone homeostasis</td><td></td><td>106</td></tr><tr><td>GLS (1MOI3M)</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>Avoid self-toxicity of 10HI3M</td><td>AtIGMT5 (AT1G76790)</td><td>66</td></tr><tr><td>GLS (4MOI3M)</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>AtIGMT1 (AT1G21100), AtIGMT2 (AT1G21120)</td><td>113, 114</td></tr><tr><td>Cinnamic acids (Feruloyl CoA)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td>Drought and salt stress tolerance</td><td>AtCCoAOMT1 (AT4G34050)</td><td>111, 112</td></tr><tr><td>Hormones (MeJA)</td><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td>AtJMT (AT1G19640)</td><td>105</td></tr><tr><td>Flavonoids (Isorhamnetin)</td><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td>Reduction of solubility</td><td></td><td>18</td></tr><tr><td>Hormones (MeSA)</td><td></td><td></td><td></td><td></td><td>x</td><td></td><td></td><td>Increase of volatility</td><td>AtBSMT1 (AT3G11480)</td><td>107</td></tr><tr><td>Flavonoids (Anthocyanins)</td><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>169, 186</td></tr><tr><td>Flavonoids (Anthocyanins)</td><td></td><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td>169, 186</td></tr><tr><td>Flavonoids (Anthocyanins)</td><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>169, 186</td></tr><tr><td>Flavonoids (Anthocyanins)</td><td></td><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td></td><td>186, 187, 188</td></tr><tr><td>Flavonoids (Anthocyanins)</td><td></td><td></td><td></td><td></td><td></td><td>x</td><td></td><td></td><td></td><td>184, 191</td></tr><tr><td>Acylation (Malonylation)</td><td>Hormones (Brassinosteroids)</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td>Color intensification</td><td></td><td>195, 196</td></tr><tr><td rowspan="2">Acylation (Aromatic acylation)</td><td>Flavonoids (Saiginols)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td>Higher antioxidant activity</td><td>AtBAT1, AtPIZ, AtPIZZA (AT4G31910)</td><td>192</td></tr><tr><td>Flavonoids (Quercetin)</td><td></td><td></td><td></td><td>x</td><td></td><td></td><td>SM homeostasis</td><td>AtFPT2, AtSCPL12 (AT2G22960)</td><td>18</td></tr><tr><td rowspan="2">Acylation (Phenylacylation)</td><td>Flavonoids (Flavonols)</td><td></td><td></td><td></td><td></td><td>x</td><td></td><td>UV stress tolerance</td><td></td><td>18</td></tr><tr><td>Glucosinolates (Glucobrassicin; 13M)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td>Increase of solubility</td><td></td><td>18</td></tr><tr><td rowspan="5">Hydroxylation</td><td>Akaloids (Camalexin)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td>Higher antioxidant activity</td><td></td><td>18, 20, 62, 63, 64, 65</td></tr><tr><td>Salicylic acid</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td>10HI3M has toxic and/or deterrent effects against nematodes</td><td>CYP81F4 (AT4G37410)</td><td>66</td></tr><tr><td>Flavonoids</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td>Hormone homesotasis</td><td>S5H (DMR6; AT5G24530); S3H (AT4G10500)</td><td>69, 71</td></tr><tr><td>Hormones (ABA)</td><td></td><td></td><td>x</td><td></td><td></td><td></td><td>UV tolerance</td><td></td><td>72, 73, 74</td></tr><tr><td>Flavonoids</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Drought, cold, heat stress response</td><td>CYP707A1 (AT4G19230); CYP707A2; CYP707A3 (AT5G45340); CYP707A4 (ABA80Xs)</td><td>20, 77</td></tr><tr><td></td><td>Coumarins</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Divalent metal chelation and homeostasis</td><td></td><td>20, 81, 84</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>x</td><td></td><td>Fe2+ Chelation, Iron accumulation</td><td></td><td>82, 83</td></tr></tbody></table>

PENETRATION2 (PEN2)-dependent breakdown products of 4MOI3M play a role in plant defence against fungal pathogens ¹¹⁴. The enzymes responsible for 4MOI3M biosynthesis from 4-hydroxyindol-3-ylmethyl glucosinolate (4OHI3M), namely indole-glucosinolate O-MT 1 (IGMT1) and indole-glucosinolate O-MT 2 (IGMT2), are induced in *A. thaliana* upon fungi infection (Figure 2). This is paralleled by the accumulation of the corresponding 4MOI3M metabolite and enhanced resistance to the pathogens ¹¹³. Interestingly, Tao and colleagues (2022)¹¹³ suggested that due to the loss of camalexin biosynthesis during evolution in Brassica crops, the pathway producing 4MOI3M is crucial for *Alternaria brassissicola* resistance in Chinese kale (*Brassica oleracea* var. *alboglabra* Bailey). Differently, *igmt5* mutants, impaired in 1-methoxyindol-3-ylmethyl glucosinolate (1MOI3M) production from the O-methylation of 1-hydroxyindol-3-ylmethyl glucosinolate (10HI3M), displayed an enhanced resistance against the root-knot nematode *Meloidogyne javanica* ⁶⁶. The authors suggest that 10HI3M and/or its derivatives have toxic and/or deterrent effects on nematodes and that its methylation could serve to avoid self-toxicity ⁶⁶. Hence, these observations highlight that depending on the position of the functional group that is methylated within a molecule, the effect of methylation on metabolite bioactivity in response to stress can be considerably different. Interestingly, GLS profiles are highly contrasted between Brassicaceae species. For instance, GLS profiles of *B. rapa*, *B. napus* and *B. juncea*, but not *B. oleracea*, include 4MOI3M and 4OHI3M ¹¹⁵. Also, more generally, *A. thaliana* has short-chain aliphatic GLS (C3 to C8 aliphatic GLS) and benzyl GLS (Figure 2) whereas *Capsella rubella*, *Capsella bursa-pastoris*, *Capsella grandiflora*, *C. sativa* and

*Neslia paniculata* do not present benzyl GLS and both long and short-chain aliphatic GLS (C3 to C10 aliphatic GLS)¹¹⁶.

## 2.3. Glycosylation

Glycosylation is a reaction involving the addition of a sugar moiety (e.g. glucose, galactose, rhamnose, arabinose or xylose), or glycan, to a SM or other organic molecules. A saccharide is transferred from a glycosyl donor molecule to a functional group, usually a hydroxyl group, from another molecule (the glycosyl acceptor).

### 2.3.1. Enzymes involved in SM glycosylation: glycosyltransferases and glucosidases

Glycosylation reaction is catalysed by glycosyltransferases enzymes (GTs; EC 2.4.1.x). Inversely, glycoside hydrolases (GHs; EC 3.2.1.x), also known as glycosidases, are involved in the hydrolysis of glycosidic bonds linking sugar moieties to other molecules. Comprehensive genomic, structural and biochemical information on GTs and glycosidases families are described in the Carbohydrate-Active enZymes Database (CAZy database - http://www.cazy.org/) ¹¹⁷.

GT enzymes catalyse the transfer of sugar moieties from donor molecules to an acceptor molecule such as saccharides, lipids, proteins, nucleic acids, or other organic compounds such as SMs ¹¹⁸,¹¹⁹. GTs are classified according to different criteria including their
phylogeny, substrate specificity, reaction mechanism, and 3D structures (GT-A, GT-B or predicted GT-C and type of reaction 102,119,120). According to these parameters, GTs are grouped in 114 families (GT1-GT114), with GT1 family being the largest family found in plants (http://www.cazy.org/) 117,118,121. SM glycosylation is usually mediated by GT1 enzymes (See Supplementary Figure S1 for A. thaliana GT1 phylogenetic tree) 30,118. Nevertheless, GTs from other families can also be involved in the glycosylation of these metabolites 118. Enzymes belonging to GT1 family usually used uridine 5-diphosphate (UDP) sugars as donor molecules and are therefore referred as UDP-glycosyltransferases (UGTs) 20,30,119. These enzymes have a C-terminal consensus sequence of 44-amino acids, defined as plant secondary product glycosyltransferase (PSPG) box 20,30,119,121. Inversely, the N-terminal fragment is more variable, suggesting its involvement in binding distinct sugar acceptors 119. UDP-glucose (C6) is the most common sugar donor for GT1 enzymes, but also UDP-galactose (C6), UDP-rhamnose (C6), UDP-arabinose (C5), UDP-xylose (C5) and UDP-glucuronic acid (C6) can also serve as donors 30,119. These UDP-sugars are transferred to -COOH, -NH2, -OH, C-C and -SH functional groups of the acceptor molecules 30,119. Currently, 112 GT1 sequence from A. thaliana (Brassicaceae) are indexed in CAZy database (http://www.cazy.org/). Moreover, 107 GT1 genes have been predicted using the PSPG motif as a search tool on A. thaliana genome 30. Those GTs are involved in glucosinolate, flavonoid, terpenoid and cinnamic acid decorations and/or biosynthesis but also in the glycosylation of xenobiotics (Figure 2, 3, 4) (Supplementary Table S1). Interestingly, substrate specificities can be distinguished between GT families. UGT78Ds are for instance specific to flavonoids whereas UGT72Es and UGT84As are characteristic to cinnamic acids and UGT71s use terpenoids as substrate (Supplementary Table S1)21,30,122–126. Hence, together with GHs, GTs determine the levels of glycosylation of SMs impacting in turns their activities 30.

### 2.3.2. Structural diversity and functions of glycosylated SMs

Glycosylations occur in most SMs classes and has significant effects on SM physicochemical properties, which affect their solubility, cellular localization, bioactivity and functions (Table 1). 30. Indeed, glycosylations increase water solubility of a wide range of metabolites 4,18. Interestingly, the position within the molecule, the type (e.g. glucose, rhamnose, arabinose, galactose) and the number of sugars (e.g. mono-, di-, tri- glycosylated) added have different effects on aglycone properties 18. For instance, the water solubility of the flavonoid aglycone quercetin and some of its glycosylated forms is as follows: quercetin-3-O-hexose-rhamnose (rutin) (3.54 mg/mL) > quercetin-3-O-rhamnose (isoquercitrin) (1.95 mg/mL) > quercetin (26 mg/ml) 18. Inversely, the redox potential of flavonoids usually decreases with glycosylation 127. As an example, rutin redox potential is of 275 mV while quercetin is of 398 mV 18. Moreover, glycosylation is known to impact transport and cellular localization of SM. As an example, phenolic compounds are accumulated in vacuoles mostly in their glycosylated forms as reviewed in detail by 128. A. thaliana TT12 (TRANSPARENT TESTA 12) MATE (multidrug and compound

extrusion) transporter 129, has been suggested to act has a flavonoid/H⁺-antiporter which transported glycosylated flavan-3-ols and anthocyanins to vacuoles 130. Besides, ABCC2 (ATP BINDING CASSETTE C2) have been biochemically found to transport several glycosylated flavonoids into vacuoles 131. Similarly, phenolic compounds such as glycosylated lignin oligomers and several lignin precursors, including glycosylated hydroxycinnamic acid derivatives, are stored in vacuoles 132. Moreover, coumarins, which are involved in plant-microbe interaction and iron acquisition 83,133,134, are also accumulated under their glycosylated forms in vacuoles 135. Under Fe deficiency, coumarin aglycones secreted to the rhizosphere by the ATP-BINDING CASSETTE G37/PLEIOTROPIC DRUGRESISTANCE9 (ABCG37/PDR9) transporter could facilitate Fe (III) availability to the FRO2 reductase to generate Fe (II) 82,83,136. Indeed, in many cases, glycosylation can inactivate bioactive aglycone compounds and facilitate their storage making them easy to be remobilized by plant when needed 18,30. This homeostasis is essential for plants to have an optimal balance between defence and growth. Several compounds are indeed crucial for plant to face biotic and biotic stresses, yet these compounds often present selftoxicity towards plant cells too. Hence, glycosylation allows to avoid this toxicity while being a modification that can be rapidly reversed by the action of glycoside hydrolases (GHs), also named glycosidases. In addition, glycosylated SMs being more stable than the corresponding aglycones, a function of UGT proteins in stress resistance may consist in increasing the stability of SMs such as phenylpropanoids 18 hence improving plant responses against oxidative damages triggered by various environmental stresses 18,137. Beside this, an improved bioactivity of glycosylated phenylpropanoids cannot be ruled out 137. In other cases, glycosylation is indeed required to increase SM bioactivity. Interestingly, in these cases complex glycosylation patterns are usually observed 30.

Hence, a high number of GTs glycosylating several SMs involved in biotic or abiotic stress response have been identified to date. An interesting study combining comparative genomic and transcriptomic analysis of GT1 genes have been recently conducted on A. thaliana, B. rapa, B. oleracea and B. napus species and allowed to identify several biotic stress-responsive GTs 121. UGT73B3, UGT73B4, and UGT73B5 were significantly upregulated in Arabidopsis in response to all the pathogens used in the analysis, including both bacteria and fungi such as *Alternaria brassicicola*, *Pseudomonas syringae* and *Hyaloperonospora arabidopsis* 121. UGT71C3, UGT74E2, and UGT85A1 genes were strongly upregulated in Arabidopsis rosette leaves infected with A. brassicicola and *Blumeria graminis*, whereas UGT73B3 and UGT89C1 were upregulated upon *Golovinomyces orontii* infection, and UGT73D1 was upregulated in leaves following *P. syringae* pv. phaseolicola infection 121. According to phylogenetic analysis, GTs from UGT73 subfamily could be also involved in pathogen response in the three Brassica species 121. UGT73B3 and UGT73B5 had already been described as involved in the hypersensitive response in an SA- depending manner, with *ugt73b73* and *ugt73b5* mutants displaying a loss of resistance to an avirulent *P. syringae* strain 138. Moreover,
UGT74F2 and UGT76B1 have been characterized in *P. syringae* response. The first is known to use isoleucic acid (2-hydroxy-3-methyl-pentanoic acid) as substrate, while both SA and N-hydroxy-pipecolic acid have been proposed as substrates of the latter ¹³⁹,¹⁴⁰. Interestingly, a role in the cross-talk between JA and SA was suggested for both of these enzymes ¹³⁹,¹⁴¹. Indeed, *UGT76B1* loss of function mutant showed enhanced resistance to the biotrophic pathogen *P. syringae* compared to wild-type plants ¹³⁹. This resistance was associated to elevated SA levels, and an increase of susceptibility towards the necrotrophic pathogen *A. brassicicola*, which is related to JA signalling ¹³⁹. Moreover, N-hydroxy-pipecolic was also found to contribute to plant enhanced resistance during *Pseudomonas* infection and the mechanism of glycosylation mediated by UGT76B1 was determined as crucial for balancing growth and defence ¹⁴⁰,¹⁴². Similarly, *UGT74F2* loss of function mutant led to enhanced resistance to the biotrophic pathogen *P. syringae* while the loss of function of the close relative *UGT74F1* gene decreased the susceptibility to the same bacterial pathogen ¹⁴¹. These were only some few examples in which UGT involvement in stress response had been identified, mainly based on mutant phenotypes.

Yet, in some cases glycosylation is known to impact SM biological activity but the GT catalysing such glycosylations is still unknown. For instance, glycosylation of camalexin is known to reduce its toxicity ⁶⁹,⁷¹. Several camalexin derivatives, including different glycosylated forms have been detected in *A. thaliana* ¹⁴³. However, no plant associated GT involved in the production of these derivatives have been characterized. Interestingly, the fungal pathogen *Sclerotinia sclerotiorum* has been found to be able to glycosylate camalexin to form non-toxic N and 6-glycosylcamalexin, hence counteracting the phytoalexin toxicity ¹⁴⁴. Moreover, glycosylated derivatives of the indole GLS 1-hydroxyindol-3-ylmethyl glucosinolate (1OHI3M) would be involved in Arabidopsis resistance against root-knot nematode *Meloidogyne javanica* ⁶⁶. Finally, glucosinolates constitute another type of SM which play crucial roles in Brassicaceae biotic stresses defences. Indeed, these compounds are characteristic of the species from this family and in some cases act as major SM involved in defence against pathogens since camalexin biosynthesis has disappeared during several Brassica crop selections/evolutions. GSL are characterized by a β-thioglycoside N-hydroxysulfate core structure, comprising a β-D glucopyranosyl moiety and a side-chain (or “R-group”) that can be aromatic, indolic, or aliphatic derivatives of amino acids precursors (Figures 1 and 2) ¹⁴. Upon fungal infection or herbivory stress, the GSL stored in vacuoles are hydrolysed by myrosinases, which are thioglycoside glucohydrolases. The removal of the β-D glucopyranosyl moiety generate unstable intermediates that are rapidly transformed in an array of bioactive compounds, such as isothiocyanates, thiocyanates or nitriles (Figure 2) ¹⁴,⁶⁶.

Several UGTs have been characterized to play a role in stresses responses in *A. thaliana*. For instance, UGT74E2 is involved in water stress response, whereas UGT85A5 and UGT75D1 play a role in salt tolerance ¹⁴⁵⁻¹⁴⁸. Moreover, UGT84A1, UGT84A2, UGT84A3 and

UGT84A4 are involved in UV stress tolerance, and a role in oxidative stress responses has been suggested for UGT73B5 and UGT73B2 ¹⁴⁹,¹⁵⁰. Phylogenetic analyses combined with genomic and transcriptomic data allowed the identification of several UGTs for which the expression is modulated by abiotic stresses in *A. thaliana* and Brassica species ¹²¹. Among them, many UGTs play a putative role in salt stress tolerance (*UGT71B1*, *UGT71C1*, *UGT71C2*, *UGT71D1*, *UGT72B2*, *UGT74D1*, *UGT74F2*, *UGT76E9*, *UGT84A2*, and *UGT84A4*), cold resistance (*UGT73C1*, *UGT78D3*, *UGT79B2*, *UGT84A3*, and *UGT90A1*), drought tolerance (*UGT73B3*, *UGT73B5*, *UGT73C1*, *UGT74E2*, and *UGT79B1*), heat tolerance (*UGT79B2*, *UGT81A1*, *UGT85A1*, *UGT85A2*, *UGT86A2*, and *UGT90A1*) and oxidative stress response (*UGT71B1*, *UGT71B2*, *UGT71B7*, *UGT74D1*, *UGT76E1*, *UGT76F2*, *UGT78D1*, *UGT87A1*, *UGT88A1*, *UGT73B3* and *UGT73B5*) ¹²¹. Although specific enzymatic activities and substrates of these enzymes are still unknown, some of them have been characterized. UGT71C1, UGT78D1, UGT78D3, UGT79B1, UGT79B2, UGT84A2 and UGT84A3 mainly use phenylpropanoids as substrates ¹²⁶,¹⁵¹⁻¹⁵⁴. Consistently, a large part of the Brassicaceae flavonoids are found under glycosylated forms, O-glycosides being the most common form in these species ²⁰. Kaempferol glycosides are the most abundant flavonols from both *A. thaliana* leaves and seeds ³,¹⁵⁵⁻¹⁵⁷. Many glycosylated flavonoids are accumulated upon UV exposure, including quercetin glycoside ¹⁵⁸ and rice flavones ¹⁵⁹. Anthocyanin accumulation has been proposed to confer abiotic stress tolerance by enhancing antioxidant capacities in *A. thaliana*. Enzymes catalysing their glycosylation, such as UGT79B2 and UGT79B3 rhamnosyltransferases acting on both cyanidin and cyanidin 3-O-glucoside, stabilize anthocyanin accumulation, and hence promote ROS scavenging capacities allowing stress tolerance ¹⁶⁰. Hormones constitute another category of metabolites subjected to glycosylation. Indeed, among the previously stress-related UGTs, UGT74F2 uses SA as substrate, whereas UGT85A1 and UGT75D1 use the cytokinin (CK) zeatin and the auxin indole-3-butyric acid (IBA), respectively, as substrates ¹⁴⁷,¹⁶¹. Finally, some GTs are also involved in the detoxification of xenobiotics, which consists of exogenous compounds harmful for plants such as herbicides, insecticides or other chemical pollutants. In *A. thaliana*, both UGT73C1 and UGT73B4 GTs can detoxify the explosive 2,4,6-trinitrotoluene (TNT) through the formation of glycosylated conjugates ¹⁶². The authors suggested that TNT conjugates were afterwards incorporated into the cell wall matrix and that sequestration within the vacuole could also take place ¹⁶². Moreover, UGT72B1 has been found to detoxify the pollutant 3,4-dichloroaniline (DCA) from *A. thaliana* root cell cultures by allowing DCA glycosylated conjugate transport to the culture medium. The purified recombinant protein displays high glycosyl transferase activity towards other xenobiotics such as 2,4,5-trichloro-phenol, chlorinated phenols ¹⁶³.

## 2.4. Acylation

Acylation is a common chemical reaction involving the transfer of an acyl group, (i.e. a carbonyl group (C=O) attached to an alkyl or aryl),
via an activated donor/energy-rich molecule to an acceptor molecule
¹⁶⁴,¹⁶⁵.

2.4.1. Enzymes involved in SM acylation: BAHD acyltransferases and SCPL acyltransferases

Acylation reactions are catalysed by acyltransferases (ATs; EC 2.3.1.x), which are ubiquitous enzymes of plants, animals, and microorganisms ¹⁶⁵,¹⁶⁶. Until now, 141 plant ATs have been characterized ¹⁶⁵. Two distinct ATs families mediate acylation in plants: BAHD-acyltransferases (BAHD-ATs) and serine carboxypeptidase-like acyltransferases (SCPL-ATs) ²⁰,¹⁶⁴,¹⁶⁵,¹⁶⁷. Phylogenetic trees of both *A. thaliana* ATs families are presented in Supplementary Figure S1.

BAHD acyltransferases

BAHD-ATs are a large family of acyltransferases that use acyl-CoA thioesters as 'energy-rich' donor molecule. They are named after the first biochemically characterized enzyme of the family, namely benzyl alcohol O-acetyltransferase (BEAT), anthocyanin O-hydroxycinnamoyltransferase (AHCT), anthranilate N-hydroxycinnamoyl/ benzoyltransferase (HCBT) and deacetylvindoline 4-O-acetyltransferase (DAT) ¹⁶⁴,¹⁶⁵,¹⁶⁸. Ranging from 48 to 55 kDa, BAHD-ATs are globular proteins that function as monomers ¹⁶⁴,¹⁶⁵,¹⁶⁸. BAHD-ATs proteins share low sequence identity (10-30 %) but have two mutual conserved motifs HXXXD and DFGWG ²⁰. HXXXD is directly involved in substrate specificity, whereas DFGWG, which is less conserved, probably play a structural rather than a catalytic role ²⁰,¹⁶⁴,¹⁶⁵,¹⁶⁹. Besides, the motif YFGNC may constitute a signature sequence for anthocyanin BAHD-ATs ¹⁶⁵,¹⁶⁹. The majority of BAHD-ATs are located in the cytosol but some can be found in the nucleus although they do not harbour nuclear-localization signals or transit peptides ¹⁶⁵. Among the 129 plant BAHD-ATs biochemically characterized until 2020 ¹⁶⁵, 10 BAHD have been found in Brassicaceae and mostly use flavonoids and cinnamic esters as substrates (Supplementary Table S1).

SCPL acyltransferases

SCPL-ATs are acyltransferases using 1-O-β-glucose esters as acyl
donors including: sinapoylglucose, feruloylglucose, caffeoylglucose,
p-coumaroylglucose or N-methyl anthraniloyl-O-glucose ¹⁶⁵,¹⁶⁶.
These D-glucose esters are produced by UGTs in the cytosol and later
transferred and stored into vacuoles, where they serve as substrate
for trans-acylation and trans-glycosylation reactions ¹⁷⁰. Indeed,
SCPL-ATs present a N-terminal signal peptide that target them into
the vacuole ¹⁷¹. SCPL-ATs are homologous to serine
carboxypeptidases (SCP) S10 family, which are α/β hydrolases using
a S-D-H catalytic triad to cleave the carboxyterminal peptide bonds
of their protein or peptide substrates ¹⁶⁵,¹⁶⁶,¹⁷². Despite the fact that
SCPL-ATs shared structural similarities with carboxypeptidases they
actually do not present hydrolytic activity toward peptide bonds
¹⁶⁶,¹⁷³,¹⁷⁴. It is supposed that SCP and SCPL-ATs emerged through
divergent evolution, and that SCPL-ATs enzymes have replaced the
hydrolytic activity by transferase activity during this process ¹⁶⁶,¹⁷³,¹⁷⁵.

SCPL-ATs were discovered in the early 2000s, although the
transacylation reaction from 1-O-β-glucose esters had already been
described several decades ago. Indeed, 1-O-β-glucose transacylation
reactions were already known to be involved in the biosynthesis of
several phenolic compounds such as sinapoylesters ¹⁷⁶, gallotannins
¹⁷⁷ and CGA ¹⁶⁴,¹⁷⁸. The first identified gene encoding an enzyme able
to transacylate from 1-O-β-glucose, named glucose acyl-transferase
(GAC), is involved in the biosynthesis of several isobutyryl glucose
esters in wild tomato species *Lycopersicon pennellii* ¹⁷⁹,¹⁸⁰. Since
then, 8 SCPL-ATs have been biochemically characterized in
Brassicaceae (Supplementary Table S1). A bioinformatic analysis
revealed that 51 SCPL genes are encoded by *A. thaliana* genome and
can be clustered in 3 clades: IA, IB and II ¹⁷¹. The 19 SCPL genes from
clade IA, including the GAC SCPL-AT and other sinapoyltransferases
(sinapoylcholine transferase, SCT and sinapoylmalate transferase,
SMT) already characterized prior to the analysis, were identified as
putative SCPL-ATs ¹⁸¹,¹⁸². Also, 25 SCPL genes whose function was
unknown clustered in clade II. Interestingly, the expression of several
SCPL genes is tissue specific whereas others are broadly expressed
in plant tissues ¹⁷¹. Seven putative SCPL-ATs are expressed in siliques,
containing the seeds: SCPL5, SCPL7, SCPL8, SCPL9, SCPL17, SCLPL19,
SCPL20 and SCPL21. Interestingly, SCPL19 is silique specific whereas
SCPL8 is ubiquitously expressed and the other mentioned SCPL-AT
are expressed in different tissues including the silique ¹⁷¹.

2.4.2. Structural diversity and functions of acylated SMs

Acylation can modify several SM classes and impact their activities
(Table 1). There are two main types of acylation: aromatic acylation,
which usually involves the addition of coumaroyl or sinapoyl, and
aliphatic acylation which most commonly consists in malonylation ⁴.
Interestingly, sinapoylglucose, which derives from sinapic acid,
constitutes the major acyl-donor molecule and is used by SCPL
enzymes as co-substrates to transfer sinapoyl moieties to SMs
(Figure 4) ¹⁸³. Flavonoids constitute a metabolic category which is
particularly subjected to acylation. For instance, anthocyanins are
produced from anthocyanidins through different modifications,
including notably acylation (Figure 3). Anthocyanins are a group of
colourful water soluble pigments responsible for the blue, purple,
pink, red or black colours of fruits, flowers and seeds ¹⁸⁴. They have
several important physiological and ecological functions in plants,
such as attraction of pollinators and seed dispersers, high light
irradiation protection and free radical scavenging activity ³,¹⁸⁴. To
date, all identified *A. thaliana* anthocyanin molecules are derived
from cyanidins, which constitute the predominant anthocyanidin
aglycones in this species. Interestingly, anthocyanins harbouring
synapoyl moieties have been detected in *A. thaliana* leaves, but not
in roots ¹⁸⁵. At3At1 (At1g03495), At3At1 (At1g03940), At5MAT
(At3G29590) and SAT (SCPL10; At2g23000) enzymes are involved in
the acylation of anthocyanins (Figure 3). Malonylation is thought to
be important in anthocyanin water solubility, stability, glycoside
enzymatic degradation protection and uptake into vacuoles, that
constitute their storage compartment ¹⁶⁹,¹⁸⁶. Aromatic acylation
stabilizes the structure of the molecule and intensifies the colour of
the pigment by intramolecular stacking of the anthocyanins with
polyphenols ¹⁸⁶⁻¹⁸⁸. Moreover, the number and type of acyl group seems to affect the stability of anthocyanins. Several studies have indeed reported that diacylated anthocyanins were more stable than the monoacetylated counterparts ¹⁸⁹. Besides, some evidences suggest a negative correlation between the antioxidant capacities of the acyl donor hydroxycinnamic acids (p-coumaric< ferulic<caffeic< sinapic acid) and the stabilities of the anthocyanin harbouring corresponding acyl groups ¹⁸⁴,¹⁹⁰. Moreover, the antioxidant scavenging activities of red cabbage anthocyanins against 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS⁺) was increased by acylation ¹⁸⁴,¹⁹¹. Moreover, Matsufuji et al. (2007)¹⁸⁹ demonstrated that caffeyl units displayed higher DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging activity than ferulyl or *p*-coumaryl units. Flavonols form another flavonoid category subjected to acylation, although this decoration is less frequent than in anthocyanins (Figure 3). The most studied example is the Arabidopsis Flavonol Phenylacyltransferase 2 (FPT2), an SCPL involved in the production of phenylacylated flavonols named saiginols ¹⁹². Interestingly, saiginols were detected only in organs exposed to high light stress, i.e. in flowers, leaves, stamens and siliques. Their synthesis was associated to plant tolerance to UV-B stress, being able to better absorb UV-B thanks to their added phenolic ring ¹⁹². Terpenoids constitute another SM category that can be acylated. For instance, several acylated derivatives of the triterpenoids thalianol, arabidiol and marnerol have been detected in *A. thaliana* roots ¹⁹³. Interestingly, triterpenoid derivatives were found to modulate both root microbiota and root growth of *A. thaliana* ¹⁹³,¹⁹⁴. Huang and colleagues (2019) tested the effects of several Arabidopsis root triterpenes (with different acylation degrees) towards the growth of 19 taxonomically diverse bacterial strains isolated from *A. thaliana* root microbiota. Interestingly, triterpenoids displayed distinct effects on bacteria growth. For instance, thalianin, which is formed from T9 thalianol derivative through THAA2-mediated acylation, was found to stimulate *Arenimonas* sp. bacteria (strain A388) growth. T9 did also stimulate *Arenimonas* sp. growth but the induction was limited in time. Hence, we can suggest that acylation affects the biological activity of thalianin towards this bacteria species. Interestingly thalianin, T9 and arabidin displayed similar patterns of inhibition against *Anthrobacter* sp. strain A224 growth indicating that depending on the bacteria species considered, a same SM/triterpenoid can have completely different effects ¹⁹³. Besides, acylation is known to be involved in brassinosteroids (BRs) homeostasis. Indeed, BRs, that are hormones involved in plant growth, development and stress responses, can be acylated by BAT1 (BR-related acyltransferase 1; PIZ) ¹⁹⁵⁻¹⁹⁷. Finally, xenobiotics also undergo acylation. The PMAT1 malonyltransferase is for instance involved in the malonylation of xenobiotic phenolic glucosides in Arabidopsis ¹⁹⁸.

### 3. Specialized metabolite decorations in Brassicaceae seeds

A comprehensive characterisation of the regulation, environmental plasticity and diversity of seed metabolism is of major importance to understand their adaptation to environmental changes and ecology. Brassicaceae seeds (e.g. *Camelina sativa* and *Brassica napus*) are major sources of plant-based proteins, oils, starch and mineral elements ³,¹⁸,¹⁹,²⁸. Brassicaceae seeds, including those of the model species *A. thaliana*, also constitute a large reservoir of SMs, including many phenylpropanoids and glucosinolates, with a wide range of biological functions with both beneficial and anti-nutritional (ANF) effects that remain to be studied ¹⁷,¹⁵⁶,¹⁹⁹. While in many Brassicaceae species the embryo is very likely the largest reservoir of SMs, seed coat and endosperm accumulate specific classes of SM, with important functions in seed adaptation to the environment and that determine seed quality. A comprehensive characterisation of the regulation, environmental plasticity and diversity of seed metabolism is of major importance to understand their adaptation to environmental changes and ecology. Brassicaceae seeds (e.g. *Camelina sativa* and *Brassica napus*) are major sources of plant-based proteins, oils, starch and mineral elements ³,¹⁸,¹⁹,²⁸. Brassicaceae seeds, including those of the model species *A. thaliana*, also constitute a large reservoir of SMs, including many phenylpropanoids and glucosinolates, with a wide range of biological functions with both beneficial and anti-nutritional (ANF) effects that remain to be studied ¹⁷,¹⁵⁶,¹⁹⁹. While in many Brassicaceae species the embryo is most likely the largest reservoir of SMs, seed coat and endosperm accumulate specific classes of SM, with important functions in seed adaptation to the environment and that determine seed quality.

In addition, seeds show a specific and very diverse specialized metabolome landscape compared to roots, leaves or flowers ²⁰⁰,²⁰¹. This suggested that seed SMs may play specific functions compared to metabolites accumulated in other tissues and organs. Similarly to what has been observed in leaves, roots and flowers, the large structural diversity observed among metabolite classes in seeds arise from the decorations added to their core structures ¹⁷. As described in previous paragraphs of this review, decorations influence the functions and role of SMs in vegetative tissues and in roots. In striking contrast, data are rather scarce about the impact of decorations on SM activities and functions in seeds of Brassicaceae (and other species), so that the identification of the enzymes involved represents a major challenge to be addressed.

#### 3.1. The plasticity of seed specialized metabolite decorations

A major impact of the environment on the nature of seed SM decorations was recently highlighted in the Brassicaceae species *C. sativa* grown for five consecutive years in the field ¹⁷,¹⁹⁹. In particular, several *C. sativa* genotypes have been cultivated for five consecutive years in the field, thus subjected to different environmental
conditions. This work, showed that SM belonging to the same
metabolic class, e.g. flavonols, glucosinolates and cinnamic acids,
and displaying similar accumulation patterns were also harbouring
similar decoration patterns. The authors hypothesized that the
decorations were strongly impacted by the environmental conditions
(i.e. the weather conditions of each growing and harvest season),
mainly in response to fluctuating temperatures and amount of
precipitations. For example, malonylated and acylated quercetin
derivatives, either di or tri -glycosides, were more accumulated in a
year characterized by high precipitations. Differently, the
accumulation of quercetin and/or kaempferol -mono and -tri -
glycosides harbouring galloyl or feruloyl groups increased in years
characterized by higher temperatures ¹⁷.

This work highlighted the major impact of the environmental
conditions in shaping the type of seed SM decorations, and showed
that the environment can induce the seed specialized metabolome.
Flavonols and glucosinolates were among the SM categories
characterized by high environmental plasticity, which was higher
compared to major seed reserve compounds, such as oil and
proteins. While they are essential for plant tolerance to abiotic and
biotic stresses, many seed SMs are considered as antinutritional
factors, namely toxic or with a negative effect on the organoleptic
quality of the derived products. This is the case of tannins, which can
interact with proteins and reduce the availability of amino acids ²⁸,²⁰².
Besides tannins, glucosinolates are as well considered antinutritional
SMs that negatively affected oil quality of Brassicaceae species, such
as rapeseed ²⁰³. Hence, despite being characterized by low plasticity,
availability and quality of seed proteins and oil can be indirectly
affected by the level of highly plastic SMs, such as glucosinolates and
flavonoids.

Besides their main role in plant vegetative tissues, decorations play
a key role in the regulation of seed SMs stability and activity ²⁰⁴,²⁰⁵.
Thus, abetter characterisation of the role and impact of decorations
of seed SMs will be essential to understand the impact of the
environmental conditions on seed quality.

## 3.2. The expression patterns of SM-decoration genes during Arabidopsis seed development

Embryo, endosperm, and seed coat development and maturation
include several stages ²⁰⁶⁻²⁰⁸. Embryo development encompass the
zygote formation, and globular, heart, torpedo, cotyledon and
maturation stages. Endosperm development occurs in parallel to
that of the embryo and it is important for providing nutrients to the
embryo itself. The seed coat develops from the integuments of the
ovule and its development and maturation parallel those of the
embryo and the endosperm.

While large diversity of SMs are produced in each tissue during their
development and maturation, a few information are available
regarding the decorations of these metabolites in seeds, including
the expression of genes/enzymes involved. Hence, we retrieved the
expression of genes coding for enzymes putatively involved in SM

glycosylation (GT1 glycosyltransferases), hydroxylation (CYP450 and
2-ODD hydroxylases), acylation (BAHD and SCPL acyltransferases)
and methylation (O-methyltransferases) from public available
datasets providing transcriptomic data on seeds at different
developmental stages and/or tissues ²⁰⁶,²⁰⁷ (Figure 5; Supplementary
Table S2). The majority of genes expressed in seeds and putatively
involved in SM decorations encode for hydroxylase proteins (335
genes), followed by acyltransferases (248 genes), GT1
glycosyltransferases (107 genes) and O-methyltransferases (35
genes). Surprisingly, among these genes, most showed the highest
expression at 18-19 days after pollination (post-mature green stage),
suggesting that SM decorations and, hence, SM diversity, evolve
during late step of seed development. This was true for
glycosyltransferases (GT1; (Figure 5a), hydroxylases (Figure 5b),
acyltransferases (Figure 5c) and methyltransferases (Figure 5d), for
which 44%, 49%, 25% and 26% of genes belonging to these classes
showed the highest expression at post-mature green stage,
respectively. This highlights the importance of late seed maturation
stages in shaping SMs diversity and, thus, activity, whereas most
studies focus on seed metabolite accumulation at earlier
developmental stages. In addition, genes expressed in seeds that
were already characterized or described for their role in SM
decoration (See Table 1) were identified and associated to each
decoration category (Figure 5). Many genes belonging to the same
gene family showed different expression behaviour that peaked at a
specific seed developmental stage and/or in specific tissue (Figures 5
and 6). Among them, three genes belonging to the UDP-
glycosyltransferase 72 family (UGT72). UGT72E1 and UGT72E2
(specific to seed coat) were expressed at late seed developmental
stages, while UGT72E3 showed the highest expression at pre-
globular and globular stages in the chalazal endosperm (Figure 6a).
Some members of the UGT72 family have been described for their
role in the glycosylation of monolignols/cinnamic acids and
flavonoids metabolites ²⁰⁹. In particular, UGT72E3 (AT5G26310) is
closely related to a poplar UGT previously shown to glycosylate
cinnamic acids in vitro ²¹⁰ and that affects the kinetics of lignin
deposition ²¹¹. As indicated above, most decoration-related genes
expressed in seeds belong to the hydroxylase class. AOP3 gene
(AT4G03050) belongs to this class and showed high relative
expression in Arabidopsis (Col-0 accession) seed embryo at mature
green developmental stage (Figure 6b). AOP3 enzyme is described as
a 2-oxoglutarate-dependent dioxygenase. While AOP3 is highly
expressed in seeds, it is not expressed in vegetative tissues in A.
thaliana Col-0 accession ²¹²,²¹³. Inversely, AOP2 (AT4G03060) is
expressed in leaves and not in seeds of A. thaliana Col-0 accession. It
has been demonstrated that AOP2 enzyme converts
methylsulfinylalkyl-GLS to alkenyl-GLS, while AOP3 is involved in the
conversion of methylsulfinylalkyl-GLS to hydroxyalkyl-GLS 10. It is
worth mentioning that A. thaliana Landsberg erecta (Ler) accession
showed opposite behaviour compared to Col-0, AOP2, but not AOP3,
being expressed in seeds ²¹². These two AOP genes seem to have a
contrasting seed-expression pattern in different A. thaliana
accessions. Further analyses are needed to better characterize their
biological role in seeds. Other well-known hydroxylases showed a
Expression of genes involved in specialized metabolite decorations during seed development

<figure><img src="image_6.png" /><figcaption>(a) Glycosyltransferases - GT1 (107 genes)</figcaption></figure>

**Cluster 1 (47 genes)**
AtF7RhaT (UGT89C1), UGT85A7, UGT85A5, UGT85A3, UGT85A1, UGT85A4, UGT73B4, UGT85A2, AtF3RhaT (UGT73C6), UGT76B1, UGT71B6, UGT72E1, AtA5GlcT (UGT75C1), AtFd3GlcT (UGT78D2), UGT72E2

**Cluster 2 (25 genes)**
UGT74F2, UGT74F1, UGT84A9, UGT72B1, AtF3AraT (UGT78D3), AtA3Glc:2"XylT (UGT79B1)

**Cluster 3 (8 genes)**

**Cluster 4 (27 genes)**
UGT74B1, AtF3RhaT (UGT78D1), UGT73C1, UGT75D1, UGT76C2, UGT72E3

<figure><img src="image_7.png" /><figcaption>(b) Hydroxylases (335 genes)</figcaption></figure>

**Cluster 1 (51 genes):**
CYP701A3, CYP705A12, CYP707A1, CYP714A (2), CYP71B15, CYP76C3, CYP79B3, CYP86C3, CYP88A3, CYP90C1, CYP94C1, CYP98A9, GA30x2, GSLOH, JOX1_JAO1

**Cluster 2 (164 genes):**
ACO2, ACO3, ACO4, CYP703A2, CYP705A1, CYP707A2, CYP710A (5), CYP735A (2), CYP74A1, CYP76C (3), CYP77A (2), CYP78A7, CYP79 (3), CYP81F2, CYP82G1, CYP83A1, CYP84A4, CYP86 (2), CYP89A9, CYP94B (2), CYP97B3, CYP98A (2), F6H1, JOX3_JAO1_JR21, JOX4_JAO4, S3H

**Cluster 3 (42 genes):**
CYP706A3, CYP707A4, CYP711A1, CYP72A7, CYP734A1, CYP74B2, CYP77B1, CYP83B1, CYP85A2, CYP88A4, CYP96A15, CYP97 (2), F6H2, GA20ox (2), GA3ox1

**Cluster 4 (39 genes):**
ACO1, AOP3, CYP51G1, CYP708A2, CYP716A1, CYP71A16, CYP72C1

**Cluster 5 (39 genes):**
ACO5, CYP707A3, CYP71A12, CYP73A5, CYP75B1_F3'H_TT7, CYP78A5, CYP79A2, CYP85A1, CYP86A (4), CYP90 (3), F3H_TT6, LDOX Ans_TT18

<figure><img src="image_8.png" /><figcaption>(c) Acyltransferases (248 genes)</figcaption></figure>

**Cluster 1 (63 genes):**
FPT2 (SCPL12), PMat1, THAA2 (BIA2),

**Cluster 2 (61 genes):**
At5Mat, SMT (SCPL8, SNG1)

**Cluster 3 (50 genes):**
SAT (SCPL10)

**Cluster 4 (32 genes):**
FACT, SCPL17, SCT (SCPL19, SNG2), SDT

**Cluster 5 (42 genes):**
3AT2, DCF, SHT

<figure><img src="image_9.png" /><figcaption>(d) Methyltransferases (35 genes)</figcaption></figure>

**Cluster 1 (13 genes):**
IGMT5, HOL1, BSMT1, CCoAOMT1, OMT1, GAMT2

**Cluster 2 (14 genes):**
IGMT2, IGMT1, TSM1

**Cluster 3 (13 genes):**
JMT, CCoAOMT7, GAMT1, IAMT1

<caption>**Figure 5:** Expression of genes involved in specialized metabolite decorations during seed development. a) Genes coding for glycosyltransferases. b) Genes coding for hydroxylases.</caption>

c) Genes coding for acyltransferases. d) Genes coding for methyltransferases.

seed-specific expression, such as *TT6* (*F3H*, *AT3G51240*), *TT7* (*F3'H*,
AT5G07990) and LDOX (AT4G22880), which are involved in flavonoid hydroxylation and production of flavonoid core structures ³, and showed high expression at early (globular and heart) seed developmental stages (Figure Yb). TT7 role in seed longevity has been recently characterized ¹⁵⁷. The tt7 KO mutants are strongly impaired in seed longevity, seed coat composition and seed coat development compared to wild-type plants. These phenotypes were (directly or indirectly) caused by an over accumulation of kaempferol-3-rhamnoside, which is formed from kaempferol rhamnosylation by UGT78D1 glycosyltransferase. The link between seed longevity and kaempferol-rhamnoside was further confirmed by the fact that the lack of UGT78D1 supresses the observed phenotypes in tt7 mutant.

As for the seed-expressed acyltransferase genes, many belong to the serine carboxypeptidase-like (SCPL) family (Figure 6c). Among them those coding for the sinapoylglucose: malate sinapoyltransferase (SMT; AT2G22990), sinapoylglucose:anthocyanin sinapoyltransferase (SAT; At2g23000) and sinapoylglucose:choline sinapoyltransferase (SCT, AT5G09640) enzymes can be highlighted ²¹⁴. SMT and SAT genes showed the highest expression in the seed coat at early developmental stages, while SCT was highly expressed in the embryo at mature green stage (Figure 6c). In Brassicaceae species, SMT, SAT and SCT enzymes catalysed the synthesis of sinapate esters using sinapoyl-glucose (G) as a common substrate ²¹⁴,²¹⁵. These included the conversion of sinapoyl-G to sinapoyl-

<figure><img src="image_10.png" /><figcaption>**Figure 6:** Seed tissue and expression behaviour of genes involved in SM decorations. a) Examples of glycosyltransferase expressions: UGT72s expression (UGT72E1, UGT72E2, UGT72E3). b) Examples of hydroxylase expressions: AOP3, F3H, F3'H, LDOX. c) Examples of acyltransferase expressions: SMT (SCPL8, SNG1), SAT (SCPL10), SCT (SCPL9, SNG2). d) Examples of methyltransferase expressions: IGMT1, IGMT2 and IGMT5.</figcaption></figure>
malate by the SMT or the synthesis of sinapoylcholine (sinapine) by SCT. The latter is a SM highly accumulated in seeds of many Brassicaceae model (Arabidopsis) and crop (rapeseed, camelina) species ¹⁹⁹,²¹⁶. These enzymes have been shown to be involved in sinapoyl-G esterification in vitro and/or have been characterized in *A. thaliana* vegetative tissues ²¹⁷⁻²¹⁹. Finally, SAT enzyme catalyse the sinapoylation of anthocyanins *A. thaliana* vegetative tissues ¹⁸³. Nevertheless, anthocyanins are not synthetized in the seeds of Arabidopsis and other Brassicaceae species. Hence, the substrate of this enzyme and the function of the products still have to be characterized in seeds. Among the methyltransferases genes expressed in seeds, three belong to the Indole Glucosinolate O-Methyltransferase (IGMT) family, catalysing the transfer of methyl groups for glucosinolate SMs ⁶⁶. *IGMT1* (AT1G21100) and *IGMT2* (AT1G21120) were expressed at late stage of seed maturation, and *IGMT5* (AT1G76790) showed the highest expression at mature green stage (Figure 6d).

## Future perspectives in the elucidation of plant and seed specialized metabolite decorations

The overview and analysis of the current knowledge on the diversity, activity and functions of SM decorations in Brassicaceae highlighted that SM decorations have major roles in plant biology and ecology, emphasizing the interest of their study for agroecology, food, pharmaceutical or cosmetics industrial sectors.

Brassicaceae seeds constitute a valuable model to study SM decorations and, more generally, seeds could be used as natural biofactories to produce and store a wide range of SM with a high added value. Brassicaceae family include species ranging from model plants (*Arabidopsis*) to widely used crops (e.g. rapeseed and camelina), which facilitate the laboratory study while including species widely produced over the world, especially for oil and protein production purpose. As indicated in this review, few studies focus on the elucidation of specialized metabolic decorations during seed development and in response to seed abiotic and biotic stresses. A particular attention should be given to the characterisation of the impact of decorations on the activity of antinutritional factors that are accumulated in seeds. This is the case of GLSs that are synthetized in the funiculus, constituting a main hub for GLS synthesis, and that are then transported to seeds ²²⁰,²²¹. GLS are historically considered as antinutritional factors having a negative impact of seed oil and proteins quality, and being in some cases toxic for humans. They are indeed the target of domestication in some Brassicaceae species, including Brassica and Camelina species, for which many efforts were and are being done to drastically reduce their content in the whole plant and, more recently, specifically in seeds by targeting *GTR1* and *GTR2* seed-GLS transporters, members of the nitrate/peptide (NTR/PTR) transporter family ²⁰³,²²²⁻²²⁴.

Despite being antinutritional factors, GLSs compounds are essential metabolites that mediate plant resistance to plant pathogens and bioagressors, and are over-accumulated in response to abiotic stresses. Moreover, a major role in seed defence to counteract damping-off during seed germination and seedling establishment or in pathogen resistance during seed development cannot be rule out and deserve further characterisation. More generally, the impact of the decorations (e.g. hydroxylation or acylation) on the activity of seed GLS should be better characterized and an equilibrium should be found out between the reduction of antinutritional compounds without compromising plant and seed resistance to stresses.

Although the term “decoration” of specialized metabolites is quite recent, some class of genes and enzymes performing those reactions (e.g. hydroxylases) have been studied and indexed for many years. The large production and availability of genomic, epigenomic, transcriptomic, proteomic and phosphoproteomic data will facilitate the discovery and characterisation of new genes and enzymes involved in SM decorations in the coming years. The discovery and the in-depth characterisation of the activity of new SM decoration enzymes are important for a comprehensive characterisation of their nature and functions in plants. This is especially true for the study of seed SMs, for which, compared to vegetative tissues, little is known about SM regulation under control and/or stress conditions. As suggested above, the study of SM decoration enzymes may probably result in the finding of interesting traits to select in crops. Indeed, several SMs constitute for instance alternatives to synthetic pesticides. Similarly, SM decoration enzymes constitute valuable “tools” for biotech applications.

The heterologous expression in host plants (e.g. *Nicotiana benthamiana*) or microorganisms (e.g. *Escherichia coli* or *Saccharomyces cerevisiae*) of the genes coding for those enzymes constitute a great tool in fundamental science to characterize their functions and reconstruct the related pathways, and could promote greener/eco-friendly production of already existing or new molecules. These techniques helped in the gene-pathways reconstruction and in the production of many SM of interest for pharmaceutical, cosmetic, food or agroecology sector ²²⁵⁻²²⁹.

Furthermore, as illustrated in this review, SMs play crucial roles throughout plant life cycle. A high SM number were identified and characterized but the great majority has yet to be discovered. Indeed, given that many SM pathways are restricted to certain plant order, families, genus, species or even genotypes, it is difficult to estimate the real number of natural compounds that are produced in plants. Moreover, SMs are usually produced in small amounts and, in some cases, can only be produced under certain environmental/stress conditions (e.g. oxylipin, ²³⁰⁻²³² and/or in specific organs and/or tissues (e.g. seed tannins, ¹⁵⁶. Besides, in the recent years, we are seeing a rise in the development of tools, pipelines and techniques to analyse targeted and, mostly, untargeted metabolomic data that are of great help in the reconstruction of metabolic pathways and SM annotation. Being the most accurate and precise method to determine molecule
structures, NMR (nuclear magnetic resonance) was the principal methodology used. Nevertheless, the amount of material/compound and the time needed to perform NMR analysis constitute a limitation to the use of this technique. Untargeted metabolomics constitute a field allowing the detection of a wide range of metabolic features by using Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS[MS]), Ion Chromatography coupled to tandem Mass Spectrometry (IC-MS[MS]) or Gas Chromatography coupled to tandem Mass Spectrometry (GC-MS[MS]). However, there is still some bottlenecks and limitations associated to this methodology pipeline. First, the extraction methods of SMs do not allow to extract the whole metabolome of a given sample since this depends on the polarity and stability, and/or the concentration of the SMs in the extracted tissue/fraction. Moreover, the number of available annotated SMs in databases is (still) relatively low compared to the metabolic features that can be detected by untargeted metabolomic analyses. Molecular networks (MNs), that can be generated with software such as MetGem or GNPS, constitute a powerful tool to expand the number of metabolic features associated to metabolic categories 233,234. MNs techniques allow to cluster SMs according to their chemical structure similarities, i.e. the degrees of similarity of MS² spectra. Indeed, SMs with high degree of chemical similarity share comparable MS² fragmentation pathways. Hence, MNs allow to associate a non-annotated metabolic feature (i.e. with a MS² spectra not available in databases) to a metabolic category, with a quite high level of confidence. This technique is more accurate and working better when SM standards are available. Nevertheless, to get a putative annotation of an unknown metabolic feature a closer look has to be taken to the MS² spectra. Software such as Sirius or MS2analyzer are useful to manually annotate the metabolic features based on their MS² spectra 235,236. However, the use of those software, dealing with complex data, could be improved. The settlement of a proper database including the characteristic product ions and fragments associated to the functional groups/decorations that can occur on the different SM structures, similarly to the list if major plant metabolite mass spectrometry diagnostic fragments that have been proposed by Wang et al. (2019)⁴, appears necessary and crucial to improve and facilitate the annotation of SMs. A promising strategy to annotate and characterize metabolic pathways in model and non-model plants and seeds could come from the combination of single-cell (sc) untargeted metabolomics with sc-transcriptomics and genomic techniques. This strategy has been recently adopted to characterize the monoterpene indole alkaloids biosynthesis in the non-model medicinal plant *Catharanthus roseus* 237.

All together these new emerging strategies and pipelines constitute a great tool to finely elucidate pathways, genes and/or enzymes involved in SM production, and to characterize the function and activity of the produced metabolites.

## Funding

The IJPB benefits from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007). The Ph.D. scholarship of L.B. is funded by the 'Plant biology and breeding' (BAP) department of INRAE and by the LabEx Saclay Plant Sciences-SPS. Part of the work presented in this review is supported by the 'Plant biology and breeding' (BAP) department of INRAE (grant 'Appel à projets scientifiques BAP 2020' to M.C.), by the project 'Seed MetSpe' of the Labex Saclay Plant Sciences-SPS (AN-10-LABX-0040-SPS to L.L. and M.C.) and by a project financed by AgroParisTech ('Appel à projets scientifiques 2020' to L.R. and M.C.). Part of the results presented in this review has been produced by the IJPB's Plant Observatory technological platforms.

## Supplementary data

The supplementary tables S1 and S2 have been deposited at the INRAE data repository portal (data INRAE) with the following identifier: https://doi.org/10.57745/ZIEUVQ

## Data availability

The transcriptomic data of *A. thaliana* seeds collected at different developmental stages are available as supplementary material in the following article: Belmonte et al. 2013. *PNAS* 110 (5): E435–44.
https://doi.org/10.1073/pnas.1222061110.

The transcriptomic data of different *A. thaliana* seed tissues and collected at different developmental stages are available at the "BAR eFP Browser" integrated in the "The Arabidopsis Information Resource" (TAIR) website, and published as supplementary material in the following article: Le et al. 2010. *PNAS* 107 (18): 8063–70.
https://doi.org/10.1073/pnas.1003530107.

## Conflicts of interest

The authors declare that they have no competing interests.

## References

1. S. Bhatla and M. Lal, *Plant Physiology, Development and Metabolism*, Springer Nature Singapore, 2018.

2. M. Erb and D. J. Kliebenstein, *Plant Physiol.*, 2020, **184**, 39–52.

3. M. Corso, F. Perreau, G. Mouille and L. Lepiniec, *Plant Sci.*, 2020, **296**, 110471.

4. S. Wang, S. Alseekh, A. R. Fernie and J. Luo, *Mol. Plant*, 2019, **12**, 899–919.

5. E. Pichersky and E. Lewinsohn, *Annu. Rev. Plant Biol.*, 2011, **62**, 549–566.

6. C. S. Westfall, A. M. Muehler and J. M. Jez, *J. Biol. Chem.*, 2013, **288**, 19304–19311.
7 D. A. T. Boncan, S. S. K. Tsang, C. Li, I. H. T. Lee, H. Lam, T. Chan and J. H. L. Hui, *Int. J. Mol. Sci.*, 2020, **21**, 7382.

8 V. Ninkuu, L. Zhang, J. Yan, Z. Fu, T. Yang and H. Zeng, *Int. J. Biol. Macromol.*

9 S. D. Tetali, *Planta*, 2019, **249**, 1–8.

10 I. I. Abdallah and W. J. Quax, *KnE Life Sci.*, 2017, **2016**, 81–98.

11 T. D. Nguyen and T. T. T. Dang, *Front. Plant Sci.*, DOI:10.3389/fpls.2021.682181.

12 J. W. Fahey, A. T. Zalcmann and P. Talalay, *Phytochemistry*, 2001, **56**, 5–51.

13 R. Mithen, R. Bennett and J. Marquez, *Phytochemistry*, 2010, **71**, 2074–2086.

14 I. Blažević, S. Montaut, F. Burčul, C. E. Olsen, M. Burow, P. Rollin and N. Agerbirk, *Phytochemistry*, 2020, **169**, 112100.

15 D. B. Clarke, *Anal. Methods*, 2010, **2**, 310–325.

16 S. R. Bhandari, J. S. Jo and J. G. Lee, *Molecules*, 2015, **20**, 15827–15841.

17 S. Boutet, L. Barreda, F. Perreau, J. Totozafy, C. Mauve, B. Gakière, E. Delannoy, M. Martin-Magniette, A. Monti, L. Lepiniec, F. Zanetti and M. Corso, *Plant J.*, 2022, 147–165.

18 M. Corso, F. Perreau, L. Rajjou, R. Ben Malek, L. Lepiniec and G. Mouille, in *Advances in Botanical Research*, 2021, pp. 1–36.

19 L. Lepiniec, I. Debeaujon, J. M. Routaboul, A. Baudry, L. Pourcel, N. Nesi and M. Caboche, *Annu. Rev. Plant Biol.*, 2006, **57**, 405–430.

20 S. Alseekh, L. Perez de Souza, M. Benina and A. R. Fernie, *Phytochemistry*, DOI:10.1016/j.phytochem.2020.112347.

21 T. Tohge, L. P. De Souza and A. R. Fernie, *J. Exp. Bot.*, 2017, **68**, 4013–4028.

22 N. Shen, T. Wang, Q. Gan, S. Liu, L. Wang and B. Jin, *Food Chem.*, 2022, **383**, 132531.

23 The 1001 Genomes Consortium, *Cell*, 2016, **2**, 481–491.

24 T. Kawakatsu, S. C. Huang, F. Jupe, E. Sasaki, J. Schmitz, M. A. Urich, R. Castanon, J. R. Nery, C. Barragan, H. Chen, M. Dubin, C. Lee, C. Wang, F. Bemm, C. Becker, R. O. Neil, R. C. O. Malley and D. X. Quarless, *Cell*, 2016, **166**, 492–505.

25 M. Togninalli, J. A. Freudenthal, J. G. Monroe, D. Meng, M. Nordborg, D. Weigel, K. Borgwardt, A. Korte and G. Grimm, *Nucleic Acids Res.*, 2020, **48**, 1063–1068.

26 A. C. A. L. Campos, F. A. William, P. Ramakrishna, T. Giles, P. Korte, A. Douglas, P. Smith and D. E. Salt, *Plant J.*, 2021, **106**, 536–554.

27 D. Raimondi, M. Corso, P. Fariselli and Y. Moreau, *Nucleic Acids Res.*, 2022, **50**, e16.

28 S. Baud, M. Corso, I. Debeaujon, B. Dubreucq, D. Job, A. Marion-Poll, M. Miquel, H. North and L. Lepiniec, *Comptes Rendus. Biol.*, 2023, **354**, 61–110.

29 S. Desmet, K. Morreel and R. Dauwe, *Plants*, 2021, **10**, 2393.

30 T. Louveau and A. Osbourn, *Cold Spring Harb. Lab. Press*, 2019, **11**, a034744.

31 V. D'Amelia, T. Docimo, C. Crocoll and M. M. Rigano, *Genes (Basel)*, 2021, **12**, 936.

32 D. Cox-Georgian, N. Ramadoss, C. Dona and C. Basu, *Med. plants from farm to Pharm.*, 2019, **Chapter 15**, 333–359.

33 M. Hartmann, T. Zeier, F. Bernsdorff, T. Hölzel, C. Ganter, M. Hartmann, T. Zeier, F. Bernsdorff, V. Reichel-Deland, D. Kim, M. Hohmann, S. Nicola, S. Stefan, B. Andrea and Z. Jürgen, *Cell*, 2018, **173**, 456–469.

34 R. Eggers, A. Jammer, S. Jha, B. Kerschbaumer, M. Lahham, E. Strandback, M. Toplak, S. Wallner, A. Winkler and P. Macheroux, *Phytochemistry*, 2021, **189**, 112822.

35 C. C. Hansen, D. R. Nelson, B. L. Møller and D. Werck-Reichhart, *Mol. Plant*, 2021, **14**, 1244–1265.

36 D. R. Nelson, *BBA - Proteins Proteomics*, 2018, **1866**, 141–154.

37 N. Laffaru Singpho and J. . Sharma, *IOP Conf. Ser. earth Environ. Sci.*, 2021, **775**, 012012.

38 J. Xu, X. Y. Wang and W. Z. Guo, *J. Integr. Agric.*, 2015, **14**, 1673–1686.

39 M. Klingenberg, *Arch. Biochem. Biophys.*, 1958, **75**, 376–386.

40 T. Omura and R. Sato, *J. Biol. Chem.*, 1964, **239**, 2370–2378.

41 R. A. Mckinnon, M. J. Sorich and M. B. Ward, *J. Pharm. Pract. Res.*, 2008, **38**, 55–57.

42 F. P. Guengerich, *Chem. Res. Toxicol.*, 2001, **14**, 611–650.

43 B. Meunier, P. De Visser and S. Shaik, *Chem. Rev.*, 2004, **104**, 3947–3980.

44 B. A. Pandian, R. Sathishraj, M. Djanaguiraman, P. V. V. Prasad and M. Jugulam, *Antioxidants*, 2020, **9**, 454.

45 H. Wang, Q. Wang, Y. Liu, X. Liao, H. Chu, H. Chang, Y. Cao, Z. Li, T. Zhang, J. Cheng and H. Jiang, *Synth. Syst. Biotechnol.*, 2021, **6**, 102–109.

46 P. Chakraborty, A. Biswas, S. Dey, T. Bhattacharjee and S. Chakrabarty, *J. Xenobiotics*, 2023, **13**, 402–423.
47 F. Durst and D. P. O. Keefe, *Drug Metabol. Drug Interact.*, 1995, **12**, 171-188.

48 D. R. Nelson, R. Ming, M. Alam and M. A. Schuler, *Trop. Plant Biol.*, 2008, **1**, 216-235.

49 J. Yu, S. Tehrim, L. Wang, K. Dossa, X. Zhang, T. Ke and B. Liao, *BMC Genomics*, 2017, **18**, 733.

50 M. Pfalz, H. Vogel and J. Kroymann, *Plant Cell*, 2009, **21**, 985-999.

51 M. D. Mikkelsen, C. H. Hansen, U. Wittstock and B. A. Halkier, *J. Biol. Chem.*, 2000, **275**, 33712-33717.

52 C. Wang, M. M. Dissing, N. Agerbirk, C. Crocoll and B. A. Halkier, *Front. Plant Sci.*, 2020, **11**, 57.

53 J. Rajniak, R. F. H. Giehl, E. Chang, I. Murgia, N. Von Wirén and E. S. Sattely, *Nat. Chem. Biol.*, 2018, **14**, 442-450.

54 J. He, Q. Chen, P. Xin, J. Yuan, Y. Ma, X. Wang, M. Xu, J. Chu, R. J. Peters and G. Wang, *Nat. Plants*, 2019, **5**, 1057-1065.

55 S. Wei, W. Zhang and R. Fu, *BMC Genomics*, 2021, **22**, 126.

56 S. C. Farrow and P. J. Facchini, *Front. Plant Sci.*, 2014, **5**, 524.

57 J. M. Hagel and P. J. Facchini, *Nat. Prod. Rep.*, 2018, **35**, 721-734.

58 S. Islam, T. M. Leissing, R. Chowdhury, R. J. Hopkinson and C. J. Schofield, *Annu. Rev. Biochem.*, 2018, **87**, 585-620.

59 C. Q. Herr and R. P. Hausinger, *Trends Biochem. Sci.*, 2018, **43**, 517-532.

60 E. L. Hegg and L. J. Que, *Eur. J. Biochem.*, 1997, **250**, 625-629.

61 Y. Kawai, E. Ono and M. Mizutani, *Plant J.*, 2014, **78**, 328-343.

62 J. Treml and K. Smejkal, *Compr. Rev. Food Sci. Food Saf.*, 2016, **15**, 720-738.

63 Y. Liu, J. Qian, J. Li, M. Xing, D. Grierson, C. Sun, C. Xu, X. Li and K. Chen, *Hortic. Res.*, 2022, **9**, uhab068.

64 S. R. Husain, J. Cillard and P. Cillard, *Phytochemistry*, 1987, **26**, 2489-2491.

65 J. Mierziak, K. Kostyn and A. Kulma, *Molecules*, 2014, **19**, 16240-16265.

66 M. Pfalz, M. Mukhaimar, F. Perreau, J. Kirk, C. Ida, C. Hansen, C. E. Olsen, N. Agerbirk and J. Kroymann, *Plant Physiol.*, 2016, **172**, 2190-2203.

67 M. S. C. Pedras, E. E. Yaya and E. Glawischnig, *Nat. Prod. Rep.*, 2011, **28**, 1381-1405.

68 A. Koprivova, M. Schwier, V. Volz and S. Kopriva, *J. Exp. Bot.*, 2023, **74**, 2667-2679.

69 D. J. Kliebenstein, H. C. Rowe and K. J. Denby, *Plant J.*, 2005, **44**, 25-36.

70 P. Bednarek, B. Schneider, A. Svatos, N. J. Oldham and K. Hahlbrock, *Plant Physiol.*, 2005, **138**, 1058-1070.

71 M. S. C. Pedras and A. Q. Khan, *Bioorganic Medicinal Chem. Lett.*, 1997, **7**, 2255-2260.

72 Y. Peng, J. Yang, X. Li and Y. Zhang, *Annu. Rev. Plant Biol.*, 2021, **72**, 761-791.

73 T. Zeilmaker, N. R. Ludwig, J. Elberse, M. F. Seidl, L. Berke, A. Van Doorn, R. C. Schuurink, B. Snel and G. Van den Ackerveken, *Plant J.*, 2015, **81**, 210-222.

74 K. Zhang, R. Halitschke, C. Yin, C. Liu and S. Gan, *PNAS*, 2013, **110**, 14807-14812.

75 G. Qi, J. Chen, M. Chang, H. Chen, K. Hall, J. Korin, F. Liu, D. Wang and Z. Q. Fu, *Mol. Plant*, 2018, **11**, 1427-1439.

76 T. M. Lowe-Power, J. M. Jacobs, F. Ailloud, B. Fochs, P. Prior and C. Allen, *MBio*, 2016, **7**, e00656-16.

77 G. Agati, E. Azzarello, S. Pollastri and M. Tattini, *Plant Sci.*, 2012, **196**, 67-76.

78 K. N. Baron, D. F. Schroeder and C. Stasolla, *Plant Sci.*, 2012, **188-189**, 48-59.

79 Z. Chan, *Genomics*, 2012, **100**, 110-115.

80 M. Corso and V. Garcia de la Torre, *Metallomics*, 2020, **12**, 840-859.

81 U. A. Hartwig, C. A. Maxwell, C. M. Joseph and D. A. Phillips, *Plant Physiol.*, 1990, **92**, 116-122.

82 P. Fourcroy, N. Tissot, F. Gaymard, J.-F. Briat and C. Dubos, *Mol. Plant*, 2016, **9**, 485-488.

83 P. Fourcroy, P. Siso-Terraza, D. Sudre, M. Saviron, G. Reyt, F. Gaymard, A. Anunciacion, J. Abadia, A. Alvarez-Fernandez and J.-F. Briat, *New Phytol.*, 2014, **201**, 155-167.

84 A. M. Verdan, H. C. Wang, C. R. García, W. P. Henry and J. L. Brumaghim, *J. Inorg. Biochem.*, 2011, **105**, 1314-1322.

85 A. Arora, M. G. Nair and G. M. Strasburg, *Free Radic. Biol. Med.*, 1998, **24**, 1355-1363.

86 L. Dall'Osto, C. Lico, J. Alric, G. Giuliano, M. Havaux and R. Bassi, *BMC Plant Biol.*, 2006, **6**, 1-20.

87 G. L. Cantoni, *Annu. Rev. Biochem.*, 1975, **44**, 435-451.

88 B. A. Moffatt and E. A. Weretilnyk, *Physiol. Plant.*, 2001, **113**, 435-442.

89 S. Roje, *Phytochemistry*, 2006, **67**, 1686-1698.
90 M. Rahikainen, S. Alegre, A. Trotta, J. Pascual and S. Kangasjärvi, *Physiol. Plant.*, 2018, **162**, 162–176.

91 D. K. Liscombe, G. V. Louie and J. P. Noel, *Nat. Prod. Rep.*, 2012, **29**, 1238–1250.

92 J. W. Schmidberger, S. Andrews, A. B. James, R. Edwards, S. Andrews and D. O. Hagan, *Angew. Chemie Int. Ed.*, 2010, **49**, 3646–3648.

93 S. Balasubramani, S. Lv, Q. Chen, Z. Zhou, D. S. Moorthy, D. Sathish, A. K. Moola and D. Ph, *Plant Gene*, 2021, **27**, 100295 Contents.

94 B. Kim, Y. Lee, H. Hur, Y. Lim and J. Ahn, *Phytochemistry*, 2006, **67**, 387–394.

95 K. C. Lam, R. K. Ibrahim, B. Behdad and S. Dayanandan, *Genome*, 2007, **50**, 1001–1013.

96 Y. Liu, A. R. Fernie and T. Tohge, *Plants*, 2022, **11**, 564.

97 J. S. Morris and P. J. Facchini, *Front. Plant Sci.*, 2019, **10**, 1058.

98 I. K. Ragai, *Trends Plant Sci.*

99 S. Provenzano, C. Spelt, S. Hosokawa, N. Nakamura, F. Brugliera, L. Demelis, D. P. Geerke, A. Schubert, Y. Tanaka, F. Quattrocchio and R. Koes, *Plant Physiol.*, 2014, **165**, 962–977.

100 L. Wen, Y. Jiang, J. Yang, Y. Zhao, M. Tian and B. Yang, *Ann. N. Y. Acad. Sci.*, 2017, **1398**, 120–129.

101 D. Weiss, A. van der Luit, E. Knegt, E. Vermeer, J. N. M. Mol and J. M. Kooter, *Plant Physiol.*, 1995, **107**, 695–702.

102 P. M. Coutinho, E. Deleury, G. J. Davies and B. Henrissat, *J. Mol. Biol.*, 2003, **328**, 307–317.

103 M. Varbanova, S. Yamaguchi, Y. Yang, K. McKelvey, A. Hanada, R. Borochov, F. Yu, Y. Jikumaru, J. Ross, D. Cortes, C. Ma, J. P. Noel, L. Mander, V. Shulaev, Y. Kamiya, S. Rodermel, D. Weiss and E. Pichersky, *Plant Cell*, 2007, **19**, 32–45.

104 Y. Yang, R. Xu, C. Ma, A. C. Vlot, D. F. Klessig and E. Pichersky, *Plant Physiol.*, 2008, **147**, 1034–1045.

105 S. Fonseca, J. M. Chico and R. Solano, *Curr. Opin. Plant Biol.*, 2009, **12**, 539–547.

106 S. Abe, A. Sado, K. Tanaka, T. Kisugi, K. Asami, S. Ota, H. Il, K. Yoneyama, X. Xie, T. Ohnishi, Y. Seto, S. Yamaguchi, K. Akiyama, K. Yoneyama and T. Nomura, *PNAS*, 2014, **111**, 18084–18089 | .

107 F. Chen, J. C. D. Auria, D. Tholl, J. R. Ross, J. Gershenzon, J. P. Noel and E. Pichersky, *Plant J.*, 2003, **36**, 577–588.

108 J. Ryals, S. Uknes and E. Ward, *Plant Physiol.*, 1994, **104**, 1109–112.

109 V. Shulaev, P. Silverman and N. Brunswick, *Nature*, 1997, **385**, 718–721.

110 S. Park, E. Kaimoyo, D. Kumar, S. Mosher and D. F. Klessig, *Science (80-).*, 2008, **318**, 113–116.

111 H. J. Chun, D. Baek, H. M. Cho, S. H. Lee, B. J. Jin, D. Yun, Y. Hong and M. C. Kim, *Plant Signal. Behav.*, 2019, **14**, e1625697.

112 H. J. Chun, L. H. Lim, M. S. Cheong, D. Baek, M. S. Park, H. M. Cho, S. H. Lee, B. J. Jin, D. H. No, Y. J. Cha, Y. B. Lee, J. C. Hong, D. Yun and M. C. Kim, *Plants*, 2021, **10**, 831.

113 H. Tao, H. Miao, L. Chen, M. Wang, C. Xia, W. Zeng, B. Sun, F. Zhang, S. Zhang, C. Li and W. Qiaomei, *J. Integr. Plant Biol.*, 2022, **64**, 1007–1019.

114 P. Bednarek, M. Pislewska-Bendarek, A. Svatos, B. Schneider, J. Doubsky, M. Masurova, M. Humphry, C. Consonni, R. Panstruga, A. Sanchez-Vallet, A. Molina and P. Schulze-Lefert, *Science (80-).*, 2009, **323**, 101–106.

115 A. P. Essoh, F. Monteiro, A. R. Pena, M. S. Pais, M. Moura and M. M. Romeiras, *Plant Physiol. Biochem.*, 2020, **150**, 151–161.

116 P. Czerniawski, A. Piasecka and P. Bednarek, *Phytochemistry*, DOI:10.1016/j.phytochem.2020.112571.

117 E. Drula, M. L. Garron, S. Dogan, V. Lombard, B. Henrissat and N. Terrapon, *Nucleic Acids Res.*, 2022, **50**, D571–D577.

118 J. Lao, A. Oikawa, J. R. Bromley, P. McInerney, A. Suttangkakul, A. M. Smith-Moritz, H. Plahar, T. Y. Chiu, S. M. González Fernández-Niño, B. Ebert, F. Yang, K. M. Christiansen, S. F. Hansen, S. Stonebloom, P. D. Adams, P. C. Ronald, N. J. Hillson, M. Z. Hadi, M. E. Vega-Sánchez, D. Loqué, H. V. Scheller and J. L. Heazlewood, *Plant J.*, 2014, **79**, 517–529.

119 P. Tiwari, R. S. Sangwan and N. S. Sangwan, *Biotechnol. Adv.*, 2016, **34**, 714–739.

120 J. A. Campbell, G. J. Davies, V. Bulone and B. Henrissat, *Biochem. J.*, 1997, **326**, 929.

121 H. M. Rehman, M. A. Nawaz, Z. H. Shah, J. Ludwig-Müller, G. Chung, M. Q. Ahmad, S. H. Yang and S. I. Lee, *Sci. Rep.*, 2018, **8**, 1875.

122 D. M. Priest, R. G. Jackson, D. A. Ashford, S. R. Abrams and D. J. Bowles, *FEBS Lett.*, 2005, **579**, 4454–4458.

123 Z. Liu, J. Yan, D.-K. Li, Q. Luo, Q. Yan, Z.-B. Liu, L.-M. Ye, J.-M. Wang, X.-F. Li and Y. Yang, *Plant Physiol.*, 2015, **167**, 1659–1670.

124 A. Lanot, D. Hodge, R. G. Jackson, G. L. George, L. Elias, E. Lim and D. J. Bowles, *Plant J.*, 2006, **48**, 286–295.

125 T. Tohge, K. Matsui, M. Ohme-takagi, M. Yamazaki and K. Saito, *Biotechnol. Lett.*, 2005, **27**, 297–303.
126 K. Yonekura-Sakakibara, T. Tohge, F. Matsuda, R. Nakabayashi, H. Takayama, R. Niida, A. Watanabetakahashi, E. Inoue and K. Saito, *Plant Cell*, 2008, **20**, 2160–2176.

127 M. Plaza, T. Pozzo, J. Liu, K. Zubaida, G. Ara, C. Turner and E. N. Karlsson, *J. Agric. Food Chem.*, 2014, **62**, 3321–3333.

128 N. Shitan and K. Yazaki, *BBA - Biomembr.*, 2020, **1862**, 183127.

129 I. Debeaujon, A. J. M. Peeters, K. M. Léon-kloosterziel and M. Koornneef, *Plant Cell*, 2001, **13**, 853–871.

130 K. Marinova, L. Pourcel, B. Weder, M. Schwarz, D. Barron, J. M. Routaboul, I. Debeaujon and M. Kleina, *Plant Cell*, 2007, **19**, 2023–2038.

131 C. E. Behrens, K. E. Smith, C. V Iancu, J. Choe and J. V Dean, *Sci. Rep.*, 2019, **9**, 437.

132 O. Dima, K. Morreel, B. Vanholme, H. Kim, J. Ralph and W. Boerjan, *Plant Cell*, 2015, **27**, 695–710.

133 S. Clemens and M. Weber, *Plant Signal. Behav.*, 2016, **11**, e1114197 (6).

134 N. B. Schmid, R. F. H. Giehl, S. Döll, H. Mock, N. Strehmel, D. Scheel, X. Kong, R. C. Hider and N. Von Wirén, *Plant Physiol.*, 2014, **164**, 160–172.

135 G. Taguchi, S. Fujikawa, T. Yazawa, R. Kodaira, N. Hayashida, M. Shimosaka and M. Okazaki, *Plant Sci.*, 2000, **151**, 153–161.

136 K. Robe, F. Gao, L. Lefebvre-Legendre, E. Sylvestre-Gonon, S. Hem, N. Rouhier, M. Barberon, A. Hecker, E. Izquierdo and C. Dubos, *New Phytol.*, 2021, **229**, 2062–2079.

137 M. Behr, G. Neutelings, M. El Jaziri and M. Baucher, *Front. Plant Sci.*, 2020, **11**, 571399.

138 M. Langlois-Meurinne, C. M. M. Gachon and P. Saindrenan, *Plant Physiol.*, 2005, **139**, 1890–1901.

139 V. von Saint Paul, W. Zhang, B. Kanawati, B. Geist, T. Faus-Keßler, P. Schmitt-Kopplin and A. R. Schaffner, *Plant Cell*, 2011, **23**, 4124–4145.

140 L. Mohnike, D. Rekhter, W. Huang, K. Feussner, H. Tian, C. Herrfurth, Y. Zhang and I. Feussner, *Plant Cell*, 2021, **33**, 735–749.

141 B. Boachon, J. Gamir, V. Pastor, M. Erb, J. V Dean, V. Flors and B. Mauch-Mani, *Eur. J. plant Pathol.*, 2014, 707–720.

142 L. Mohnike, W. Huang, B. Worbs, K. Feussner, Y. Zhang and I. Feussner, *J. Exp. Bot.*, 2023, **74**, 458–471.

143 C. Bottcher, L. Westphal, C. Schmotz, E. Prade, D. Scheel and E. Glawischnig, *Plant Cell*, 2009, **21**, 1830–1845.

144 M. Soledade, C. Pedras and P. W. K. Ahiahonu, *Biorgan. Med. Chem.*, 2002, **10**, 3307–3312.

145 V. B. Tognetti, O. Van Aken, K. Moreel, K. Vandenbroucke, B. van de Cotte, I. De Clercq, S. Chiwocha, R. Fenske, E. Prinsen, W. Boerjan, B. Genty, K. A. Stubbs, D. Inze and F. Van Breusegem, *Plant Cell*, 2010, **22**, 2660–2679.

146 J. T. Song, Y. J. Koo, H. S. Seo, M. C. Kim, Y. Do Choi and J. H. Kim, *Phytochemistry*, 2008, **69**, 1128–1134.

147 G.-Z. Zhang, S.-H. Jin, X.-Y. Jiang, R.-R. Dong, P. Li, Y.-J. Li and B.-K. Hou, *Plant Mol. Biol.*, 2016, **90**, 77–93.

148 Y.-G. Sun, B. Wang, S.-H. Jin, X.-X. Qu, Y.-J. Li and B.-K. Hou, *PLoS One*, 2013, **8**, e59924.

149 A. Mazel and A. Levine, *Plant Physiol. Biochem.*, 2002, **40**, 133–140.

150 D. Meißner, A. Albert, C. Böttcher, D. Strack and C. Milkowski, *Planta*, 2008, **228**, 663–674.

151 P. Jones, B. Messner, J.-I. Nakajima, A. R. Schäffner and K. Saito, *J. Biol. Chem.*, 2003, **278**, 43910–43918.

152 E. K. Lim, Y. Li, A. Parr, R. Jackson, D. A. Ashford and D. J. Bowles, *J. Biol. Chem.*, 2001, **276**, 4344–4349.

153 K. Yonekura-Sakakibara, A. Fukushima, R. Nakabayashi, K. Hanada, F. Matsuda, S. Sugawara, E. Inoue, T. Kuromori, T. Ito, K. Shinozaki, B. Wangwattana, M. Yamazaki and K. Saito, *Plant J.*, 2012, **69**, 154–167.

154 E. Lim, G. S. Higgins, Y. Li and D. J. Bowles, *Biochem. J.*, 2003, **373**, 987–992.

155 J. C. D'Auria and J. Gershenzon, *Curr. Opin. Biotechnol.*, 2005, **8**, 308–316.

156 J. Routaboul, L. Kerhoas, I. Debeaujon, L. Pourcel, M. Caboche, J. Einhorn and L. Lepiniec, *Planta*, 2006, **224**, 96–107.

157 R. Ninoles, P. Arjona, S. M. Azad, A. Hashim, J. Casan, E. Bueso, R. Serrano, A. Espinosa, I. Molina and J. Gadea, *New Phytol.*, 2023, **238**, 1461–1478.

158 K. G. Ryan, E. E. Swinny, C. Winefieldb and K. R. Markham, *Zeitschrift für Naturforsch. C*, 2001, **56**, 745–754.

159 M. Peng, R. Shahzad, A. Gul, H. Subthain, S. Shen, L. Lei, Z. Zheng, J. Zhou, D. Lu, S. Wang, E. Nishawy, X. Liu, T. Tohge, A. R. Fernie and J. Luo, *Nat. Commun.*, 2017, **8**, 1975.

160 P. Li, Y.-J. Li, F.-J. Zhang, G.-Z. Zhang, X.-Y. Jiang, H.-M. Yu and B.-K. Hou, *Plant J.*, 2017, **89**, 85–103.

161 S.-H. Jin, X.-M. Ma, M. Kojima, H. Sakakibara, Y.-W. Wang and B.-K. Hou, *Planta*, 2013, **237**, 991–999.

162 F. Gandia-Herrero, A. Lorenz, T. Larson, I. A. Graham, D. J. Bowles, E. L. Rylott and N. C. Bruce, *Plant J.*, 2008, **56**, 963–974.

163 C. Loutre, D. P. Dixon, M. Brazier, M. Slater, D. J. Cole and R. Edwards, *Plant J.*, 2003, **34**, 485–493.
164 T. Bontpart, V. Cheynier, A. Ageorges and N. Terrier, *New Phytol.*, 2015, **208**, 695–707.

165 L. Wang, K. Chen, M. Zhang, M. Ye and X. Qiao, *Crit. Rev. Biotechnol.*, 2021, **42**, 125–144.

166 A. Ciarkowska, M. Ostrowski, E. Starzyńska and A. Jakubowska, *Phytochem. Rev.*, 2019, **18**, 303–316.

167 X. Yan, X. Qin, W. Li, D. Liang, J. Qiao and Y. Li, *Planta*, 2020, **252**, 1–12.

168 J. C. D'Auria, *Curr. Opin. Plant Biol.*, 2006, **9**, 331–340.

169 T. Nakayama, H. Suzuki and T. Nishino, *J. Mol. Catal. B Enzym.*, 2003, **23**, 117–132.

170 N. Sasaki, Y. Nishizaki, Y. Ozeki and T. Miyahara, *Molecules*, 2014, **19**, 18747–18766.

171 C. M. Fraser, L. W. Rider and C. Chapple, *Plant Physiol.*, 2005, **138**, 1136–1148.

172 T. Bontpart, M. Ferrero, F. Khater, T. Marlin, S. Vialet, A. Vallverdù-Queralt, L. Pinasseau, A. Ageorges, V. Cheynier and N. Terrier, *Plant Physiol. Biochem.*, 2018, **130**, 356–366.

173 C. Milkowski and D. Strack, *Phytochemistry*, 2004, **65**, 517–524.

174 F. Stehle, W. Brandt, M. T. Stubbs, C. Milkowski and D. Strack, *Phytochemistry*, 2009, **70**, 1652–1662.

175 F. Stehle, W. Brandt, C. Milkowski and D. Strack, *FEBS Lett.*, 2006, **580**, 6366–6374.

176 B. Dahlbender and D. Strack, *Planta*, 1984, **161**, 142–147.

177 G. G. Gross, *Zeitschrift für Naturforsch. C*, 1983, **38**, 519–523.

178 M. Kojima and I. Uritani, *Plant Physiol.*, 1973, **51**, 768–771.

179 A. X. Li, N. Eannetta, G. S. Ghangas and J. C. Steffens, *Plant Physiol.*, 1999, **121**, 453–460.

180 A. X. Li and J. C. Steffens, *PNAS*, 2000, **97**, 6902–6907.

181 C. Lehfeldt, A. M. Shirley, K. Meyer, M. O. Ruegger, J. C. Cusumano, P. V Viitanen, D. Strack and C. Chapple, *Plant Cell*, 2000, **12**, 1295–1306.

182 A. M. Shirley and C. Chapple, *J. Biol. Chem.*, 2003, **278**, 19870–19877.

183 C. M. Fraser, M. G. Thompson, A. M. Shirley, J. Ralph, J. A. Schoenherr, T. Sinlapadech, M. C. Hall and C. Chapple, *Plant Physiol.*, 2007, **144**, 1986–1999.

184 N. Zhang and P. Jing, *Crit. Rev. Food Sci. Nutr.*, 2022, **62**, 2205–2220.

185 M.-Z. Shi and D.-Y. Xie, *Recent Pat. Biotechnol.*, 2014, **8**, 47–60.

186 H. Suzuki, T. Nakayama, K. Yonekura-Sakakibara, Y. Fukui, N. Nakamura, M. Yamaguchi, Y. Tanaka, T. Kusumi and T. Nishino, *Plant Physiol.*, 2002, **130**, 2142–2151.

187 T. Goto and T. Kondo, *Angew. Chemie Int. Ed. English*, 1991, **30**, 17–33.

188 R. Brouillard and O. Dangles, *Food Chem.*, 1994, **51**, 365–371.

189 H. Matsufuji, H. Kido, H. Misawa, J. Yaguchi, T. Otsuki, M. Chino, M. Takeda and K. Yamagata, *J. Agric. Food Chem.*, 2007, **55**, 3692–3701.

190 F. Natella, M. Nardini, M. Di Felice and C. Scaccini, *J. Agric. Food Chem.*, 1999, **47**, 1453–1459.

191 W. Wiczkowski, D. Szawara-nowak and J. Topolska, *Food Res. Int.*, 2013, **51**, 303–309.

192 T. Tohge, R. Wendenburg, H. Ishihara, R. Nakabayashi, M. Watanabe, R. Sulpice, R. Hoefgen, H. Takayama, K. Saito, M. Stitt and A. R. Fernie, *Nat. Commun.*, 2016, **7**, 12399.

193 A. C. Huang, T. Jiang, Y.-X. Liu, Y.-C. Bai, J. Reed, B. Qu, A. Goossens, H.-W. Nutzmann, Y. Bai and A. Osbourn, *Plant Sci.*, 2019, **364**, eaau6389.

194 Y. Bai, P. Fern, A. C. Huang, S. Morales-, K. U. Bicalho, M. Karady, L. Pauwels, D. Buyst, C. Martins, S. Vanneste, T. Beeckman, A. Osbourn, K. Ljung, A. Goossens and J. Pollier, *New Phytol.*, 2020, **230**, 228–243.

195 S. Choi, Y.-H. Cho, K. Kim, M. Matsui, S.-H. Son, S.-K. Kim, S. Fujioka and I. Hwang, *Plant J.*, 2013, **73**, 380–391.

196 K. Schneider, C. Breuer, A. Kawamura, Y. Jikumaru, A. Hanada, S. Fujioka, T. Ichikawa, Y. Kondou, M. Matsui, Y. Kamiya, S. Yamaguchi and K. Sugimoto, *PLoS One*, 2012, **7**, e46805.

197 T. M. Nolan, N. Vukasinovic, D. Liu, E. Russinova and Y. Yin, *Plant Cell*, 2020, **32**, 295–318.

198 G. Taguchi, T. Ubukata, H. Nozue, Y. Kobayashi, M. Takahi, H. Yamamoto and N. Hayashida, *Plant J.*, 2010, **63**, 1031–1041.

199 B. Alberghini, F. Zanetti, M. Corso, S. Boutet, L. Lepiniec, A. Vecchi and A. Monti, *Ind. Crops Prod.*, 2022, **182**, 114944.

200 F. Matsuda, M. Y. Hirai, E. Sasaki, K. Akiyama, K. Yonekura-Sakakibara, N. J. Provart, T. Sakurai, Y. Shimada and K. Saito, *Plant Physiol.*, 2010, **152**, 566–578.

201 C. Yang, S. Shen, S. Zhou, Y. Li, Y. Mao, J. Zhou, Y. Shi, L. An, Q. Zhou, W. Peng, Y. Lyu, X. Liu, W. Chen, S. Wang, L. Qu, X. Liu, A. R. Fernie and J. Luo, *Mol. Plant*, 2022, **15**, 258–275.

202 N. Nesi, R. Delourme, M. Brégeon, C. Falentin and M. Renard, *C.R. Biol.*, 2008, **331**, 763–771.
203 H. H. Nour-Eldin, S. R. Madsen, S. Engelen, M. E. Jørgensen, C. E. Olsen, J. S. Andersen, D. Seynnaeve, T. Verhoye, R. Fulawka, P. Denolf and B. A. Halkier, *Nat. Biotechnol.*, 2017, **35**, 377-382.

204 J. Luo, C. Fuell, A. Parr, L. Hill, P. Bailey, K. Elliott, S. A. Fairhurst, C. Martin and A. J. Michael, *Plant Cell Plant Cell*, 2009, **21**, 318-333.

205 K. Clauß, E. von Roepenack-Lahaye, C. Böttcher, M. R. Roth, R. Welti, A. Erban, J. Kopka, D. Scheel, C. Milkowski and D. Strack, *Plant Physiol.*, 2011, **155**, 1127-1145.

206 B. H. Le, C. Cheng, A. Q. Bui, J. A. Wagmaister, K. F. Henry, J. Pelletier, L. Kwong, M. Belmonte, R. Kirkbride, S. Horvath, G. N. Drews, R. L. Fischer, J. K. Okamuro, J. J. Harada and R. B. Goldberg, *Proc. Natl. Acad. Sci.*, 2010, **107**, 8063-8070.

207 M. F. Belmonte, R. C. Kirkbride, S. L. Stone, J. M. Pelletier, A. Q. Bui, E. C. Yeung, M. Hashimoto, J. Fei, C. M. Harada, M. D. Munoz, B. H. Le, G. N. Drews, S. M. Brad, R. B. Goldberg and J. J. Harada, *Proc. Natl. Acad. Sci.*, 2013, **110**, E435-E444.

208 S. Baud, J. P. Boutin, M. Miquel, L. Lepiniec and C. Rochat, *Plant Physiol. Biochem.*, 2002, **40**, 151-160.

209 N. Speeckaert, M. El Jaziri, M. Baucher and M. Behr, *Biology (Basel)*, 2022, **11**, 441.

210 N. Speeckaert, N. M. Adamou, H. A. Hassane, F. Baldacci-cresp, A. Mol, G. Goeminne, W. Boerjan, P. Duez, S. Hawkins, G. Neutelings, T. Ho, W. Schwab, M. El Jaziri, M. Behr and M. Baucher, *Int. J. Mol. Sci.*, 2020, **21**, 5018.

211 F. Baldacci-Cresp, J. Le Roy, B. Huss, C. Lion, A. Créach, C. Spriet, L. Duponchel, C. Biot, M. Baucher, S. Hawkins and G. Neutelings, *Int. J. Mol. Sci.*, 2020, **21**, 6094.

212 D. J. Kliebenstein, V. M. Lambrix, M. Reichelt, J. Gershenzon and T. Mitchell-Olds, *Plant Cell*, 2001, **13**, 681-693.

213 L. M. Jensen, H. S. K. Jepsen, B. A. Halkier, D. J. Kliebenstein and M. Burow, *Front. Plant Sci.*, 2015, **6**, 697.

214 A. M. Shirley, C. M. McMichael and C. Chapple, *Plant J.*, 2001, **28**, 83-94.

215 C. M. Fraser and C. Chapple, *Arab. Book/American Soc. Plant Biol.*, 2011, **9**, e0152.

216 C. Milkowski and D. Strack, *Planta*, 2010, **232**, 19-35.

217 F. Stehle, M. T. Stubbs, D. Strack and C. Milkowski, *FEBS J.*, 2008, **275**, 775-787.

218 F. Stehle, W. Brandt, J. Schmidt, C. Milkowski and D. Strack, *Phytochemistry*, 2008, **69**, 1826-1831.

219 X. Li, J. Bergelson and C. Chapple, *Mol. Plant*, 2010, **3**, 91-100.

220 D. Khan, J. L. Millar, I. J. Girard, A. Chan, R. C. Kirkbride, J. M. Pelletier, S. Kost, M. G. Becker, E. C. Yeung, C. Stasolla, R. B. Goldberg, J. J. Harada and M. F. Belmonte, *Plant J.*, 2015, **82**, 41-53.

221 D. Xu, N. Christian, H. Sanden, L. L. Hansen, Z. M. Belew, S. R. Madsen, L. Meyer, M. E. Jørgensen, P. Hunziker, D. Veres, C. Crocoll, A. Schulz, H. H. Nour-eldin and B. A. Halkier, *Nature*, 2023, **617**, 1-7.

222 D. M. Nambiar, P. K. Bajpai, J. Kumari, R. Augustine, P. Kumar, P. K. Bajpai and N. C. Bisht, *Plant Cell Environ.*, 2021, **44**, 2729-2743.

223 G. Hölzl, B. R. Rezaeva, J. Kumlehn and P. Dörmann, *Plant Biotechnol. J.*, 2023, **21**, 189-201.

224 A. Mann, J. Kumari, R. Kumar, P. Kumar, A. K. Pradhan, D. Pental and N. C. Bisht, *Plant Biotechnol. J.*, 2023, 1-14.

225 S. T. Mugford, X. Qi, S. Bakht, L. Hill, E. Wegel, R. K. Hughes, K. Papadopoulou, R. Melton, M. Philo, F. Sainsbury, G. P. Lomonosoff, A. D. Roy, R. J. M. Goss and A. Osbourn, *Plant Cell*, 2009, **21**, 2473-2484.

226 V. Courdavault, S. E. O'Connor, M. K. Jensen and N. Papon, *Nat. Prod. Rep.*, 2021, **38**, 2145-2153.

227 B. Hong, D. Grzech, L. Caputi, P. Sonawane, C. E. R. López, M. O. Kamileen, N. J. H. Lozada, V. Grabe and S. E. O. Connor, *Nature*, 2022, **607**, 617-622.

228 J. Zhang, L. G. Hansen, O. Gudich, K. Viehrig, L. M. M. Lassen, L. Schrübbers, K. B. Adhikari, P. Rubaszka, E. Carrasquer-alvarez, L. Chen, V. D. Ambrosio, B. Lehka, A. K. Haidar, S. Nallapareddy, K. Giannakou, M. Laloux, D. Arsovska, M. A. K. Jørgensen, L. J. G. Chan, M. Kristensen, H. B. Christensen, S. Sudarsan, E. A. Stander, E. Baidoo, C. J. Petzold, T. Wulff, S. E. O. Connor, V. Courdavault, M. K. Jensen and J. D. Keasling, *Nature*, 2022, **609**, 341-347.

229 J. Reed, A. Orme, A. El-demerdash, C. Owen, L. B. B. Martin, R. C. Misra, S. Kikuchi, M. Rejzek, A. C. Martin, A. Harkess, J. Leebens-mack, T. Louveau, M. J. Stephenson and A. Osbourn, *Science (80-.),* 2023, **379**, 1252-1264.

230 E. Lacchini, J. Venegas-molina and A. Goossens, *Curr. Opin. Plant Biol.*, 2023, **74**, 102371.

231 J. E. Jeon, J.-G. Kim, C. R. Fischer, N. Mehta, C. Dufour-Schroif, K. Wemmer, M. B. Mudgett and E. Sattely, *Cell*, 2020, **180**, 176-187.e19.

232 I. Monte, S. Kneeshaw, J. M. Franco-Zorrilla, A. Chini, A. M. Zamarreño, J. M. García-Mina and R. Solano, *Curr. Biol.*, 2020, **30**, 962-971.e3.

233 F. Olivon, N. Elie, G. Grelier, F. Roussi, M. Litaudon and D. Touboul, *Anal. Chem.*, 2018, **90**, 13900-13908.

234 M. Wang, *Nat. Biotechnol.*, 2016, **34**, 828-837.

235 Y. Ma, T. Kind, D. Yang, C. Leon and O. Fiehn, *Anal. Chem.*,
2014, **86**, 10724–10731.

236 K. Dührkop, M. Fleischauer, M. Ludwig, A. A. Aksenov, A. V Melnik, M. Meusel, P. C. Dorrestein, J. Rousu and S. Böcker, *Nat. Methods*, 2019, **16**, 299–302.

237 C. Li, J. C. Wood, A. H. Vu, J. P. Hamilton, C. Eduardo, R. Lopez, R. M. E. Payne, D. Ayled, S. Guerrero, K. Gase, K. Yamamoto, B. Vaillancourt, L. Caputi, S. E. O. Connor and C. R. Buell, *Nat. Chem. Biol.*, 2023, **19**, 1031–1041.

238 S. Harun, M. R. Abdullah-Zawawi, H. H. Goh and Z. A. Mohamed-Hussein, *J. Agric. Food Chem.*, 2020, **68**, 7281–7297.

239 S. Chhajed, I. Mostafa, Y. He, M. Abou-hashem, M. El-Domiaty and S. Chen, *Agronomy*, 2020, **10**, 1786.

240 K. Hooshmand and I. S. Fomsgaard, *Metabolites*, 2021, **11**, 47.

241 T. Saigo, T. Wang, M. Watanabe and T. Tohge, *Curr. Opin. Plant Biol.*, 2020, **55**, 93–99.

242 J. Barros, L. Escamilla-trevino, L. Song, X. Rao, J. C. Serrani-yarce, M. D. Palacios, N. Engle, F. K. Choudhury, T. J. Tschaplinski, B. J. Venables, R. Mittler and R. A. Dixon, *Nat. Commun.*, 1–11.