Survey of Genes Involved in Biosynthesis,Transport,and Signaling of Phytohormones with Focus on Solanum lycopersicum

Curation of Phytohormone Pathway Information

Genes involved in biosynthesis, transport, and signaling pathways of seven phytohormone classes for the reference model organism A. thaliana were extracted from the Arabidopsis Hormone Database version 2.0 (AHD2.0; http://ahd.cbi.pku.edu.cn/ ²⁵ ). Cross-evaluation from the AHD2.0 was performed to enzymes belonging to biosynthetic pathways extracted from the AraCyc (part of Plant Metabolic Pathways; http://pathway.gramene.org/gramene/aracyc.shtml ⁸¹ ). Next, putative candidates and complex enzyme schematic diagrams of the AHD2.0 were verified by an extensive literature screening for the seven different phytohormone classes in book chapters and reviews from the last 10 years. For enzymes assigned to be putatively involved in chemical reactions of the phytohormone pathways in Aracyc and/or AHD2.0, we searched for experimental evidence as well. A. thaliana gene identifier (AGI) and associated description was curated based on Gene Ontology (GO) information deposited in The Arabidopsis Information Resource database (TAIR10; http://www.arabidopsis.org/ ⁸² ). The identified genes were assigned to the categories biosynthesis, transport, or signaling.

Orthologue Search and Functional Domain Annotation

The plant genomes of (i) A. thaliana (TAIR10; https://www.arabidopsis.org/), (ii) Brachypodium distachyon (bradi1.2 with GAEVAL; http://www.plantgdb.org), (iii) C. reinhardtii (JGI v4 with GAEVAL; http://www.plantgdb.org), (iv) Glycine max (Glyma1; http://www.plantgdb.org), (v) Medicago truncatula (Mt3.5v5 http://jcvi.org), (vi) Oryza sativa (MSU Version 7.0 with GAEVAL; http://www.plantgdb.org), (vii) Physcomitrella patens (Phypa1.6; http://phytozome.net), (viii) Populus trichocarpa (Ptr v2.0 with GAEVAL; http://www.plantgdb.org), (ix) Solanum lycopersicum (ITAG2.4; https://solgenomics.net/organism/Solanum_lycopersicum/genome), (x) Solanum tuberosum (PGSC v3.4; http://potatogenome.net), (xi) Sorghum bicolor (JGI Sbi1; http://www.plantgdb.org), (xii) Vitis vinifera (Genescope 12X; http://genoscope.cns.fr), and (xiii) Zea mays (B73 RefGen v2; http://www.plantgdb.org), which have been downloaded from PlantGDB, ⁸³ have verified gene annotation in relation to alternative splicing and gene fusions/fissions by the gene annotation evaluation algorithm (GAEVAL 82). ⁸⁴ For the orthologue search via OrthoMCL ⁸⁵ and InParanoid, ⁸⁶ we selected these plant species to sample the tree of the super kingdom Viridiplantae by at least one species of the phylum Chlorophyta (C. reinhardtii), one species of the class Bryopsida (P. patens), and four species of the branch of monocots (S. bicolor, B. distachyon, O. sativa, and Z. mays) of which O. sativa and Z. mays are of large agricultural relevance. Besides economic importance, the seven eudicot species (A. thaliana, G. max, M. truncatula, P. trichocarpa, S. tuberosum, S. lycopersicum, and V. vinifera) were chosen as representative model organisms of distinct plant families or with broad interest in specific plant research fields (Fig. 1A).^87,88 G. max was chosen due to agricultural relevance and S. tuberosum and V. vinifera were chosen due to their phylogenetic relation to S. lycopersicum and comparison with previous studies. ⁸⁹

The strategy to merge results of different orthologue algorithms has been justified in Paul et al. ⁸⁷ OrthoMCL was used to determine cliques of likely orthologous genes (CLOGs) for more than two species, and results were verified using the pairwise approach of InParanoid version 8. ⁸⁶ Genes were further analyzed when occurring in at least one of the two approaches. OrthoMCL filtered out poor-quality sequences based on the protein sequence length (<10 amino acids) and percentage of stop codons (marked by asterisks in the amino acid sequence; >20%). We searched in all CLOGs for all extracted genes of A. thaliana coding for enzymes involved in biosynthesis, transport, and signaling pathways of the seven phytohormone classes (373 genes based on our curation of phytohormone pathway information; Supplementary Tables 1-14).

Prediction of Domain Architecture

Domain prediction of proteins of all 13 plant species was performed using Pfam database (version 26.0), ⁹⁰ WebMGA, ⁹¹ and HMMsearch. ⁹² Customized Python scripts (www.python.org) were used to analyze domain architecture of the protein and conservation of domains of the co-orthologues in a particular orthologous group. Within one orthologous group, the domain architecture of the A. thaliana reference protein sequence was compared to the other co-orthologues concerning the order of the domains, the absence of domains, or the occurrence of new ones. The name of the PFAM domain is indicated when discussed, and the detailed description of the individual domains is available in the PFAM database (http://pfam.sanger.ac.uk/).

Prediction of Protein Localization

The prediction of the localization of identified proteins was performed by MultiLoc2, TargetP, and Predotar, which enable automated prediction by submitting ≥2 sequences at once or using local versions of the software. The predictor MultiLoc2 ⁹³ distinguishes between 11 different cellular compartments (extracellular, nucleus, Golgi, endoplasmic reticulum (ER), mitochondrion, plastid, plasma membrane, peroxisome, vacuole, cytosol, and cytoskeleton). The localization results of MultiLoc2 for vacuole, ER, Golgi, and plasma membrane are merged and represented as endomembranes. TargetP ⁹⁴ and Predotar ⁹⁵ are widely used programs and can distinguish between chloroplast, mitochondria, and secretory pathway localization and are used for a more accurate prediction of the localization of proteins in these plant compartments.

Transcriptome profiling

Existing S. lycopersicum transcriptome expression data (RNA-seq libraries; GSE33507) ⁸⁹ were obtained from the gene expression omnibus (GEO). ⁹⁶ The RNA-seq data contain S. lycopersicum (cv. Moneymaker) libraries from different organs: root, stem, leaf, flower, and fruits at three developmental stages (mature green, breaker stage, and 10 days after turning red).

General Procedure for Information Extraction

We extracted the information for the phytohormone pathways established for A. thaliana (“Methods” section) and assigned co-orthologues to the proteins involved in these pathways in 13 photosynthetic eukaryotes including Arabidopsis (Fig. 1A, Supplementary Tables 1-7). In contrast to other orthologue studies in plants such as the study by Wu et al. ⁹⁷ , our approach was not based on a single plant clade like Euasterid but attempt coverage of the whole Viridiplantae super kingdom by using model organisms from algae, mosses, monocots, and eudicots. The orthologue search was based on proteomes, and by this, the referenced tools OrthoMCL and InParanoid allowed detection of co-orthologues and putative paralogs without being restricted to single-copy orthologous genes. ⁹⁷ The approach in our study allowed the detection of CLOGs for phytohormone biosynthesis, transport, and signaling. Identified co-orthologues and co-orthologues (multiple co-orthologues of the same species) ⁹⁸ in one CLOG were analyzed for the presence of conserved functional domains found in the A. thaliana bait as it was performed in earlier studies.^88,99 Coorthologues containing all functional domains were assumed to be functionally related; however, we also considered proteins with additional or missing domains as relevant co-orthologues due to domain “stealing, swapping, or swiveling”. ¹⁰⁰ Additionally, putative targeting signals for intracellular localization were predicted to verify that the proteins within a CLOG share enzyme functionality and similar intracellular localization of the A. thaliana bait. This limited the number of co-orthologues with putative function in phytohormone pathways, because most pathways or parts of them are defined to specific organelles (Supplementary Tables 8-14). Focusing on tomato, the expression patterns of co-orthologues in the phytohormone pathways in tissues and fruit developmental stages gave hints for rate-limiting steps and tissue-specific expression. Publically available expression values in different tissues and fruit developmental stages based on “reads per kilobase per million mapped reads” (RPKM) normalized datasets were extracted from the GEO ⁹⁶ and Sol Genomics Network (SGN; Supplementary Tables 15-21). ¹⁰¹ The expression data of co-orthologues in CLOGs were categorized as low expression (RPKM < 5), moderate expression (RPKM < 100), and high expression (RPKM ≥ 100). All information are deposited in a database, ¹⁰² focusing on the assignment of the recently published tomato genome. ⁸⁹

The indole-3-pyruvic Acid Pathway is a Major, but not the only, route of auxin biosynthesis in tomato

Two major pathways of IAA biosynthesis have been proposed so far. ²⁶ The tryptophan (Trp)-independent pathway derives from the plastidial Trp-biosynthesis route, which is initiated with the conversion of chorismate to anthranilate (Fig. 1B, green region). This pathway consumes immediate precursors of Trp, ie, indole-3-glycerol phosphate or indole for the production of IAA. However, the molecular identity of the required enzyme remains unknown up to now. The Trp-dependent IAA synthesis unifies several routes of IAA biosynthesis deriving from Trp. These routes vary in the formation of distinct intermediates leading to the names of the particular pathways, ie, the indole-3-pyruvic acid (IPA) pathway (Fig. 1B, blue area), the indoleacetamine (IAM) pathway (Fig. 1B, yellow area), and the indole-3-acetaldoxime (IAOX) pathway (Fig. 1B, red area). The high number of intermediates and identified genes proposed to be involved in their enzymatic processing (Supplementary Tables 1 and 8) provides a high complexity to auxin biosynthesis concerning different possible biosynthetic pathways, which is still under debate. ²⁶

The IPA pathway includes two enzymatic reactions to convert Trp to IAA.^21,103,104 Either TAA1/TAR1 or TAR2 catalyzes the first reaction. While we could observe TAR2 co-orthologues in all plants except for green algae, TAA1/TAR1 was specific for eudicots (Fig. 1B). Remarkably, tomato co-orthologues to TAA1/TAR1 were not expressed in any of the analyzed tissues (Fig. 2). The second is catalyzed by YUC1 and YUC2, two proteins encoded by the YUCCA gene family of flavin monooxygenases. Co-orthologues of all these genes were found in all species with the exception of C. reinhardtii (Fig. 1B, Supplementary Tables 1, 8, and 15). Again, the expression of tomato YUC1, 2 co-orthologues, was low in most of the tissues (RPKM < 5) compared to other genes of the synthesis pathway (Fig. 2). This might point to conversion of IPA to indole-3-acetaldehyde (IAD) by an indole-3-caboxylase, an enzymatic activity described for IAA synthesis in plant growth-promoting rhizobacteria species, which has not been identified in plants yet. Co-orthologues of AAO1, the proposed aldehyde oxidase activity required for the subsequent conversion of IAD to IAA, were detected by our analysis in all plants, and their moderate expression in tomato exceeded that of YUC co-orthologues (RPKM > 5; Fig. 2). Nevertheless, it needs to be mentioned that broad substrate specificity was observed for the AAO1 multigene family that might link its activity to ABA synthesis as well, which is still discussed.^105,106

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Figure 2

Expression of tomato genes encoding Trp and IAA biosynthesis enzymes. Major routes and enzyme activities involved in auxin biosynthesis are represented as in Figure 1 B with focus on the expression of corresponding co-orthologues, genes identified in the tomato genome. Relative transcript levels in different tissues and developmental stages are shown in the color code as indicated in the right lower corner.

The IAM pathway also predicts two steps for the conversion of Trp to IAA with IAM as an intermediate product (Fig. 1B). The pathway resembles the conversion of Trp to IAA found in Agrobacterium strains. ¹⁰⁷ In our study, only co-orthologues of AMI1, the enzyme that catalyzes the second step,^108,109 were identified in all plants except for P. patens. AMI co-orthologues were highly expressed in tomato leaves compared to other organs (Fig. 2). In contrast, proteins similar to the bacterial proteins encoded by aux1/iaaM/tms1 genes were not identified. Recently, the conversion of IAOX to IAM was suggested as an alternative route to generate IAM. ¹¹⁰

The activity of YUCCA enzymes is assigned to the IAOX pathway for converting tryptamine (TAM) into IAOX (Fig. 1B). However, we detected neither tomato co-orthologues to A. thaliana NIT1, 2 enzymes converting tryptophan to TAM nor to enzymes converting indole-3-acetonitril (IAN) to IAA (Fig. 1B). This observation stands in line with discussion that the IAOX pathway is present in Brassicaceae only. ¹¹¹ Furthermore, the identified co-orthologues of the cytochrome P450 oxidases CYP79B2/B3 involved in IAOX production in A. thaliana ¹¹⁰ were also not expressed in the examined tissues in tomato (Fig. 2, Supplementary Table 15). This supports the current model that the IPA pathway is the major route of auxin biosynthesis in tomato. Nevertheless, we cannot exclude that several Trp-dependent auxin biosynthesis pathways may coexist and operate in different tissues. ¹⁰³

IAA Conjugation, Storage, and Degradation is Conserved among Species

The mechanism of stimulation of adventitious root formation by indol-3-butyric acid (IBA) is well established. Further, IBA is a naturally occurring IAA precursor in many plant species, which requires a peroxisomal β-oxidation activity and the IBA synthase ¹¹² for its conversion to the biologically active IAA (Fig. 1B, gray region). The compound IBA appears as reversible auxin storage form, which is transported independent of IAA. ¹¹³ In tomato, the orthologue to IBR1 and the two IBR3 co-orthologues involved in conversion of IBA to IAA were expressed in nearly all analyzed tissues at moderate levels (Fig. 2).

Both IAA and IBA are found in conjugated forms either with amino acids like alanine (Ala) or leucine (Leu) or in ester-linked conjugates with glucose. ²⁷ Co-orthologues for IAA-specific GH3 proteins were present in all plant species, except for C. reinhardtii (Fig. 1B). The conjugates either serve as hydrolyzable storage forms or play a role in IAA degradation. ²⁶ Several IAA-leucine resistant (ILR) and ILR-like (ILL) proteins, which contribute to the release of active IAA from amino acid conjugates, exist in A. thaliana and are grouped in CLOGs containing co-orthologues of all selected plant species. Tomato co-orthologues were expressed in all analyzed tissues (Fig. 2). Another ILL protein (ILL3) has been shown to be involved in auxin biosynthesis in P. euphratica, ¹¹⁴ but was not included in the CLOG of the other ILL proteins. We observed that ILL3 co-orthologues exist in monocots and eudicots only, and the corresponding tomato gene was expressed in all tissues.

Irreversible oxidation of IAA is the major target for IAA inactivation and occurs on conjugated forms as well. The accumulation of oxIAA observed after IAA application indicates that this catabolic pathway is involved in the regulation of bioactive auxin levels in plants. Finally, conversion of IAA in its methyl ester form by IAMT1 and MES17 results in a nonpolar modified form of IAA, which can probably be transported independent of auxin transporters. We observed that the required enzymes, however, were only moderately expressed in tomato (Fig. 2), and IAMT1 co-orthologues could be found only in P. patens, eudicots and rice, but not in any other of the selected monocots or C. reinhardtii (Fig. 1B, Supplementary Table 1). This might suggest that this enzyme was lost specifically in some monocots.

The directional Cellular Auxin Transport System is Specific to Multicellular Organisms

Besides long-distance phloem transport, the directed cell-to-cell transport of IAA is essential for the regulation of auxin homeostasis. ¹¹⁵ Key regulators are PIN-type auxin transport proteins (Fig. 3A), which are distributed asymmetrically along the plasma membrane. As expected, these proteins could be detected in multicellular organisms only (Fig. 3B), and most of them were not expressed in the tomato fruit (Supplementary Table 15). The polar orientated localization of the transporter changes dynamically in response to light or physical stimuli such as gravity and defines the direction and velocity of cellular auxin transport. Release of IAA into the low pH environment of the apoplast has been shown to lead to its protonation into IAAH. AUX1/LAX1 influx carriers localized at the opposite side of the next cell facilitate uptake of the apolar IAAH by the adjacent cell. In line with its function in long-distance transport, AUX1 orthologue in tomato was only moderately expressed in roots, stem, and leaves (Supplementary Table 15), while at least one LAX1 co-orthologue was moderately expressed in all tomato tissues. According to the chemiosmosis model, IAA is deprotonated and trapped in the neutral cytosolic compartment until exported by PIN proteins or other auxin transport mechanisms consisting of P-glycoproteins of the ABCB transporter family (ABCB/PGP). While most PIN proteins are plasma membrane proteins, PIN5, PIN8, and PIN-LIKE proteins are localized at the ER membrane and regulate the intracellular distribution of IAA. ¹¹⁶ Consequently, in our analysis, PIN5 and PIN8 were grouped into two distinct CLOGs containing none of the other PIN genes (PIN1-4, PIN6-7). Further, co-orthologues of PIN5 and PIN8 were found only in monocots and eudicots and tended to occur as single-copy genes (Fig. 3A, Supplementary Tables 1 and 8). With respect to their function in intracellular transport, co-orthologues to all other PINs and NRT1.1 existed in all plants, but not in C. reinhardtii, and the number of co-orthologues varied between 3 and 14 (Fig. 3B).

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Figure 3

Auxin transport and signaling pathways. (A) Survey and localization of main transporters involved in inter- and intracellular auxin transport processes. (B) Number and relationship of PIN gene family members (Supplementary Table 8) are illustrated for the 13 selected plant species (abbreviations s–e Fig. 1) by red diamonds (Pin5), green ellipses (Pin8), and blue rectangles (Pin1-4, 6, 7). (C) Principle mechanisms and factors involved in auxin signaling in plants. For further details and explanations, see text. The bar-headed dashed line in red indicates indirect suppression mechanisms.

Auxin Perception is Tightly Linked to the Regulation of Auxin-Responsive Gene

Two classes of interacting transcription factors are involved in the control of auxin-regulated gene expression (Fig. 3C^115–117). AUX/IAA transcriptional repressors were found to be present in all monocots and eudicots and were represented by a single CLOG (Supplementary Tables 1 and 8) with varying numbers of co-orthologues ranging from 5 in tomato to 15 in A. thaliana. Remarkably, one tomato orthologue was found to be highly expressed only in fruits (Solyc09g065850), while all others were not expressed in this tissue (Supplementary Table 15). AUX/IAAs generally consist of four functional domains. The “N-terminal domain I” harbors an ethylene response factor (ERF)-associated amphiphilic repressor (EAR) motif required for recruitment of TOPLESS (TPL), which is acting as a transcriptional corepressor in the absence of auxin. Interestingly, co-orthologues to TPL were identified in all analyzed plant genomes except in C. reinhardtii. For P. patens, we could identify two TPL co-orthologues but no co-orthologues to AUX/IAA (Supplementary Table 1). Domain II of AUX/IAA proteins is required for the control of their auxin-stimulated degradation, and domains III and IV participate in the formation of homo-and hetero-oligomeric complexes including interactions with members of the second class of transcription regulators, the auxin-responsive factors (ARFs). ARFs recognize and bind through their B3 DNA-binding domain-specific auxin-response elements and act as either transcriptional repressors or activators. ¹¹⁸ In A. thaliana, the ARF subfamily consists of 15 members while we identified only six ARF co-orthologues in the genome of tomato (Supplementary Table 15).

In the presence of auxin, the activation of ARFs is controlled by degradation of AUX/IAAs and release of TPL-mediated repression of DNA-binding and transcriptional activation (Fig. 3C). Degradation of AUX/IAAs is triggered by binding of auxin to one of the F-box proteins TIR1/AFB1-5, the substrate recognition subunit in the SCF^TIR/AFB E3 ubiquitin ligase complex, which per se is acting as an intracellular auxin receptor in plants. The stability of the E3 complex is regulated by modification of the scaffold subunit Cullin1 (CUL1) through covalent binding of the ubiquitin-related small protein RUB1. Our results suggest that the regulatory regime of auxin signaling can be transferred to all phototrophic eukaryotes containing co-orthologues with the exception for C. reinhardtii (Supplementary Tables 1 and 8) in which, if present, auxin signaling must be differently perceived. Although all components of the basic SCF complex were also present in the green algae co-orthologues of the TIR/AFB, F-Box proteins were not present.

ACC Synthases form a Diverse Gene Family within the Ethylene Pathway

The biosynthesis pathway of ethylene consists of only three enzymes (Fig. 4). S-adenosylmethionine is synthesized from methionine by S-adenosyl-L-methionine synthetase (SAM1) and is converted to 1-aminocyclopropane-1-carboxylate (ACC) by ACC synthase (ACS). Our orthologue search shows that each selected plant species contains at least one orthologue for SAM1. More interestingly, the A. thaliana genome encodes 12 ACS-like genes, which could be divided into three CLOGs. ACS3 is an A. thaliana-specific pseudogene with a short protein sequence, whereas ACS10 and ACS12 belonged to the same CLOG (Supplementary Table 2), and it has been shown that these aminotransferases can complement the Escherichia coli aminotransferase mutant DL39. ¹¹⁹ Thus, there are nine authentic ACS genes in the A. thaliana genome, ¹²⁰ which were grouped in one CLOG containing co-orthologues from green algae, monocots, and eudicots but without co-orthologues from the moss P. patens. In tomato, there are at least eight characterized ACS genes, ¹²¹ but we could assign 13 co-orthologues to the nine A. thaliana ACS genes involved in ethylene synthesis (Supplementary Table 16). In the last step of ethylene biosynthesis, ACC oxidase (ACO, four genes in A. thaliana) converts ACC to ethylene. Remarkably, ACO2 and ACO3 build their own CLOG without other plant co-orthologues and ACO1 and ACO4 further fell in different CLOGs. Overall, our analysis revealed six genes in the tomato genome in the three CLOGs of ACOs, which has been shown in earlier results. ¹²¹ Coherent with the importance of ethylene for fruit development, the high expression of two genes (RPKM > 1000; Solyc01g095080, Solyc05g050010) was observed during ripening (Fig. 4 ¹²¹ ). The current model considers the formation of ACC as the rate-limiting step. SAM1 and EFE/ACO co-orthologues were highly expressed in all tissues in tomato, while the ACS co-orthologues showed only moderate expression (Fig. 4; Supplementary Table 16). However, the activity of ACS is regulated by phosphorylation at protein level as it was documented for SlACS2 (Solyc01g095080). ¹²²

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Figure 4

Pathways of ethylene synthesis and signaling. (A) Enzymes and intermediate products of the ethylene production unify to methionine (blue) as precursor for ethylene (red) synthesis. The enzymatic step catalyzed by ACS is inhibited by the action of RCD1 and ETO1 or activated by MPK3 and MPK6. Further details are presented in the text. The arrows are colored according to the species in which the enzymes were found (Fig. 1A). Expression of the identified genes in tomato is shown as explained in Figure 2 (B) The components involved in ethylene signaling are represented as interaction scheme, at which activation of downstream components is indicated by green arrows and inhibition by red bar-headed lines.

The ethylene signaling cascade starts with binding of ethylene to ER-localized receptors with protein kinase activities.^50,123,124 In all monocots, eudicots, and mosses, ethylene receptors represent a gene family that is composed of ETR1, ERS1, ETR2, ERS2, and EIN4 in A. thaliana (Supplementary Tables 2 and 9). Our analysis revealed the presence of seven co-orthologues in S. lycopersicum, from which five have been previously described (SlETR1, -2, -4, -5, and Never ripe), while two have not been yet tested in tomato (SlETR6 and -7 ¹²⁵ ; Supplementary Table 16).

In contrast to the orthologue search, previous sequence analysis divided ethylene receptors into subfamily I and II. Subfamily I members tend to have higher similarity to histidine kinases, whereas subfamily II members have acquired serine kinase activities. ¹²⁶ The ethylene signaling in A. thaliana is more dependent on subfamily I members, which cannot be functionally replaced by subfamily II members. ¹²⁷ In turn, reduced expression of the subfamily II receptor genes, SlETR4 or SlETR6, in tomato results in substantially increased ethylene sensitivity. ¹²⁴ This phenotype cannot be restored to that of wild-type by overexpression of the subfamily I receptor. Thus, the subfamily II receptors might have a more important function in tomato than in A. thaliana.

In the absence of ethylene, the receptors activate the negative regulator CTR1 (constitutive triple response1), thus repressing the activity of downstream ethylene signaling components. ³⁹ In A. thaliana, the Ser/Thr protein kinase CTR1 is localized at the ER membrane due to its physical interaction with the receptors ¹²⁸ and directly phosphorylates the C-terminal domain of EIN2 in the absence of the ethylene signal. Our orthologue search detected co-orthologues of the two A. thaliana CTR1 genes only in monocots and eudicots, while in line with former studies, four co-orthologues were found in tomato (Supplementary Table 16 ¹²⁴ ).

The physical movement of EIN2 C-terminal region from the ER membrane to the nucleus allows the ethylene signal to activate the downstream transcription factors EIN3 and EIN3-like (EILs) in the nucleus. Phosphorylation at specific sites in EIN2 region regulates translocation. ³⁹ In tomato, we identified six co-orthologues to these transcription factors (EIN4/EIL1, four co-orthologues; EIL3, two co-orthologues; Supplementary Tables 2 and 9). The ethylene signal stabilizes EIN3 and EIL transcription factors, which are short-lived proteins in the absence of ethylene, and consequently activates ERFs, inducing various physiological responses. ¹²⁹

Recent advances propose a more complex route that includes feedback-regulated transcriptional networks as well as protein and mRNA turnover regulatory modules. According to this regime, ETP1 and ETP2 control EIN2 levels, whereas EBF1 and EBF2 (EIN3-binding factor) regulate the levels of EIN3 in response to the ethylene signal. ³⁹ Interestingly, the orthologous group including ETP1 and ETP2 contains 134 additional co-orthologues of A. thaliana, most of them containing both or at least one F-Box and F-Box associated (FBA_1) domains (Supplementary Table 9). Remarkably, only one representative of B. distachyon and S. tuberosum was detected in the same CLOG, which could be due to the fact that these F-Box proteins evolve very fast or they are species-specific in their primary sequence. Four orthologues were identified for EBF1 and EBF2 in tomato and at least three of them showed moderate or high expression in all tissues (Supplementary Tables 2 and 16). However, A. thaliana ETP1 and ETP2 were grouped in one CLOG containing 134 additional putative co-orthologues but only one orthologue of B. distachyon and S. tuberosum each. With respect to the high number of co-orthologues only in Arabidopsis and the low conservation within the super kingdom of Viridiplantae, the functional relation of all the 136 co-orthologues in the ethylene signaling pathway is unlikely.

Cytokinin Biosynthesis Seems to be Specific for Monocots and Eudicots

Enzymes encoded by isopentenyltransferase (IPT) genes catalyze the initial step of cytokinin biosynthesis. Earlier work based on sequence alignments by ClustalW ¹³⁰ proposed that IPTs likely evolved in plants, although similar sequences exist in some cyanobacteria. ⁴⁵ In contrast, our orthologue search approach (see “Methods” section) confirmed the occurrence of IPT genes in all monocots and eudicots, but not in mosses or algae. The only exception was tRNA-IPT (IPT9), which was detected in all selected plant species. Nevertheless, functional equivalent enzymes for IPT in species like green algae are expected, because cytokinin has been detected in the green alga Chara globularis and putative cytokinin receptors have been found in different green algae.^131,132 We identified six co-orthologues in tomato for the nine A. thaliana IPTs, from which only one showed high expression in all tissues (Supplementary Table 17). IPT utilizes adenosine tri- or diphosphate (ATP/ADP) and dimethylallyl-diphosphate (DAMPP), the reactive isomer of isopentenyl diphosphate, to synthesize N ⁶ -(Δ ² -isopentenyl) adenosine-5‘-triphosphate and -diphosphate ribonucleotides iPRTP and iPRDP, respectively (Fig. 5A ¹³³ ). Both compounds are converted subsequently into the corresponding trans-zeatin ribonucleotides (tZRTP/tZRDP) by cytokinin trans-hydroxylases, such as the cytochrome P450-dependent monooxygenases CYP735A1 and CYP735A2. Both enzymes were identified in A. thaliana and found to be conserved in monocots and eudicots. In tomato, two co-orthologues were detected in one CLOG, which showed different expression patterns in tissues and developmental stages (Supplementary Table 17). Only CYP735A1 showed moderate expression in root, flower, and mature fruit, whereas CYP735A2 was not expressed in any of these tissues. The formation of cytokinin ribonucleosides and the free bases iP and tZ has been originally assumed to be catalyzed by 5’ ribonucleotide phosphohydrolases and adenosine nucleosidases.^134,135 However, the identification of the LONELY GUY (LOG) family of enzymes in rice and A. thaliana suggests the direct conversion into the bioactive forms of the two cytokinins. Interestingly, one CLOG containing LOG1, LOG3, and LOG4 was found to be conserved in all plant species but the remaining six A. thaliana LOG genes were clustered in different CLOGs and could be divided into Arabidopsis specific (LOG2, LOG6, and LOG9), eudicot specific (LOG7), or monocot/eudicot specific (LOG5 and LOG8). In summary, we identified four co-orthologues of the LOG family in tomato (Supplementary Table 10). Remarkably, almost all co-orthologues of IPT, CYP735A, and LOG showed low expression in fruit ripening stages (Supplementary Table 17), which on the one hand suggests that cytokinins are not produced in fruits at high levels, but on the other hand, studies on the metabolite cytokinin showed that it has an unclear influence on fruit development. ¹³⁶

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Figure 5

Pathways of cytokinin synthesis and signaling. (A) The precursor of cytokinin synthesis is DMA-PP, which is derived from the MVA or MEP pathways. The intermediate products cZRMP, iPRTP/-DP, or tZRTP/-DP are converted to the final products such as cZ, iP, or tZ (red) by the action of LOG or by yet not clearly defined 5‘-ribonucleotide phosphohydrolases and adenosine nucleosidases. For further details, see text. The arrows are colored according to the species in which the enzymes were found (Fig. 1A). Expression of the identified co-orthologues in tomato is shown as explained in Figure 2 Genes coding for enzyme activities not expressed by any orthologue are colored in gray. (B) The components involved in cytokinin signaling are presented as interaction scheme.

Although found in substantial amounts, cis-zeatin (cZ) has no biological hormone activity and cannot be converted sufficiently into trans-zeatin (tZ). ¹³⁷ Moreover, the synthesis of cZ seems to be completely independent of the tZ synthesis pathway because two of the nine IPT genes in A. thaliana (IPT2 and IPT9) are specifically involved in the formation of cZRMP by utilizing AMP and DMA-PP. ⁴⁵ We were able to assign one specific tomato orthologue for both enzymes (IPT2: Solyc11g066960; IPT9: Solyc12g014190), suggesting that this pathway is also present in tomato. cZRMP is mainly derived from the cytosolic mevalonate (MVA) pathway of IPP synthesis, while the tZ pathway preferentially consumes IPP derived from the plastidial methylerythritol phosphate (MEP) pathway.

Conjugation to glucose and irreversible cleavage are the two major reactions efficiently reducing the levels of bioactive cytokinins (Fig. 5A ¹⁸ ). N-glycosyltransferases catalyze the glycosylation of nitrogen residues in the purine ring, while O-glycosylation occurs on the oxygen in the side chain of tZ or dihydrozeatin (dhZ). The latter reaction likely produces storage forms that can be rapidly reactivated by β-glucosidases. ¹³⁸ Removal of the unsaturated side chains of tZ and iP by the activity of cytokinin oxidases (CKX) leads to their irreversible inactivation and is important for the regulation of active cytokinin levels and limitation of cytokinin effects. The members of both IPT and CKX gene families show remarkably distinct expression patterns in A. thaliana and expression of several CKX genes can be stimulated upon cytokinin application. ¹³⁹ For tomato, only one orthologue of IPT5 and another of IPT9 were found to be expressed in all tissues, while transcripts of the other IPT co-orthologues were absent in most of the tissues. None of the co-orthologues of the tomato CKX gene family was expressed in all tissues, and a high variance in the expression patterns was observed as it was shown for A. thaliana in previous studies (Supplementary Table 17). ¹³⁹ Interestingly, with the exception of the CKX family, we could show that always one gene of each enzyme family of the cytokinin biosynthesis path in tomato was expressed in every tissue. In summary, for the green algae C. reinhardtii and the moss P. patens, no co-orthologues were identified for cytokinin trans-hydroxylases and glucosyltransferases, while C. reinhardtii also lacked the CKX family. Furthermore, only the tRNA-IPT (IPT9) and a small set of LOG family genes were found to be present in the unicellular algae.

Cytokinin Transport, Perception, and Signaling

tZ-ribonucleoside (tZR) is preferentially transported toward the shoot through the xylem, while the rootward transport of cytokinins occurs through the phloem. Initially, it was discussed that cytokinin receptors (AHK2, 3, and 4 in A. thaliana), with typical domain structure of membrane-bound histidine sensor kinases, represent the extracellular recognition site for cytokinin binding. Recent studies identified most of the receptor proteins in the ER membrane, which opened the discussion whether perception of cytokinins occurs in the ER lumen rather than on the cell surface.^140–142 AHK2, 3, and 4 are proposed to form heterodimeric receptor complexes upon cytokinin perception, while AHK1 is located in a distinct orthologous group, ¹⁴³ which was confirmed by our orthologue search. Cytokinin response1 (CRE1) and Woodenleg (WOL1) are allelic to AHK4 and were identified in mutant screens for cytokinin phenotypes. ¹⁴⁴ Cytokinin insensitive1 (CKI1) is one additional, more distantly related histidine kinase in A. thaliana ¹⁴⁵ ; however, its involvement in cytokinin signaling remains to be shown. In tomato, three co-orthologues were also present in the CLOG of AHK2-4 (Supplementary Table 17), which were expressed at high levels in all analyzed tissues. In contrast, for the tomato orthologue of CKI1, no expression was observed (Supplementary Table 17).

Binding of cytokinins triggers the activation of the histidine kinase domain of AHK on the cytosolic site, leading to its autophosphorylation. Components acting downstream in the phosphorelay signaling pathway include histidine-containing phosphotransfer proteins, named AHP inA. thaliana. The AHP family consists of five members (AHP1-5) and one pseudo AHP considered as a negative regulator (AHP6). We detected four co-orthologues of AHP in tomato falling in four different CLOGs. One of the four co-orthologues was expressed in all tissues (Supplementary Table 17). For the AHP4 and AHP6 co-orthologues, no expression was detected. AHP has been shown to function in the transfer of the signal to the nucleus to regulate the activity of response regulators (ARRs) acting as transcription factors. ARRs form a large family with 15 proteins in A. thaliana, which can be divided according to their domain structure into type-A AARs acting as repressors (ARR3-9, 15-17) and type-B ARRs acting as activators (ARR1-2, 10-12).^140,146,147 In tomato, six type-A AAR and five type-B AAR co-orthologues exist (Fig. 5B; Supplementary Tables 3 and 17). For ARR16 and ARR17, only a weak response to cytokinin was reported. ¹⁴⁸ These two AARs were found only in eudicots in our analysis, which could indicate redundant or newly developed functions in cytokinin response. The stability of some of the type-B AARs is controlled by their recruitment to specific SCF E3 ubiquitin ligase complexes containing one of the four F-box proteins KISS ME DEADLY (KMD1–KMD4). Two co-orthologues of KMD were detected in tomato (Fig. 5B, Supplementary Table 3).

Type-A ARRs do not possess a defined output domain, and most of them are transcriptionally induced in response to cytokinins (Supplementary Table 10). Interestingly, for the six co-orthologues in tomato, we observed that each orthologue corresponded to a pair of Arabidopsis type-A ARRs except for the group containing ARR8 and ARR9. Furthermore, in tomato type-A ARRs were expressed in all analyzed tissues with two exceptions. The orthologue of ARR5/6 was not expressed in tomato fruit, while the orthologue of ARR16/17 was only expressed in flowers and fruits (Supplementary Table 17). Altogether, the function of type-A ARRs as negative regulators of cytokinin response by interfering with the function of positive regulators in the signal transduction pathway is widely assumed. One exception so far is ARR4, which has been shown in Arabidopsis to interact positively with phytochrome B, ¹⁴⁹ and it will be interesting to see whether this particular function is conserved in tomato and other plant species as well.

Putative Rate Limitations in Biosynthesis of ABA in Tomato

ABA synthesis is initiated in plastids and derives from the metabolism of carotenoids.^17,150 Moreover, ABA-specific defects are connected to mutations in genes acting downstream of zeaxanthin synthesis. Zeatin epoxidase (ZEP), also called ABA1, catalyzes the formation of violaxanthin, and this step is reversed by the action of violaxanthin de-epoxidase (VDE; Fig. 6A). The process known as xanthophyll cycle is assumed to provide sufficient amounts of zeaxanthin for an increased photo-oxidative protection under high-light conditions. Each of the two enzymes is encoded by a single gene in Arabidopsis and has one orthologue in tomato, whereas VDE1 was not found in C. reinhardtii (Supplementary Tables 4 and 11). In contrast to ZEP1, which was at least moderately expressed in all tissues (Supplementary Table 18), VDE1 was not expressed in roots, flowers, and ripe fruits.

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Figure 6

Pathways of abscisic acid synthesis, transport, and signaling.

Formation of 9-cis-violaxanthin and 9-cis-neoxanthin via its intermediate trans-neoxanthin are the last steps occurring in plastids before cleavage by 9-cis-epoxicarotenoid dioxygenase (NCED; five genes in A. thaliana ¹⁵¹ ) and releasing of xanthoxin from the C40 precursor into the cytosol. While the enzyme-encoding genes are known for the ABA4-dependent formation (ABA4, AT1G67080) of trans-neoxanthin, the activities required for the isomerization to the 9-cis derivatives remain to be identified. Two co-orthologues of ABA4 were identified in tomato, which are globally expressed (Fig. 6A; Supplementary Table 18). The subsequent cleavage of xanthophyll by NCED is the rate-limiting step in ABA synthesis, ¹⁷ and expression of NCED is tightly regulated in response to stress and developmental signals. In contrast to previous results, ¹⁵¹ we observed a localization signal for only three (NCED 2, 3, 5) NCEDs in A. thaliana. The number of co-orthologues in the 13 plant species ranges from none in C. reinhardtii to nine in Z. mays, all containing the carotenoid oxygenase-specific domain (retinal pigment epithelial membrane (RPE) ⁶⁶ , Supplementary Tables 4 and 11). In tomato, three NCED co-orthologues were present and one appeared to be globally expressed (Fig. 6A; Supplementary Table 18).

The final two steps of ABA synthesis occur in the cytoplasm and include the conversion of xanthoxin to ABA aldehyde catalyzed by SDR1/ABA2 and the formation of the carboxyl group mainly by abscisic acid oxidase 3 (AAO3), which is one of the three AAOs in A. thaliana. ¹⁵² All three enzymes depend on a molybdenum cofactor. ¹⁵³ Consequently, a mutant of the sulfurase in A. thaliana that produces the functional cofactor was named aba3, because strong ABA deficiency phenotypes were caused due to abolishing all AAO activities. ¹⁵⁴ In tomato, we identified two co-orthologues of ABA2, six of AAO3, and one of ABA3 (Fig. 6A; Supplementary Tables 4 and 18). However, not all co-orthologues of AAO3 might be involved in ABA synthesis. On the one hand, three of them showed only moderate expression (Fig. 6A), while on the other hand, it has been shown that Arabidopsis AAO3 is the major AAO in ABA biosynthesis.^152,153

ABA catabolism plays an important role in the regulation of ABA homeostasis, and currently, two pathways have been described. ¹⁷ The first is initiated by 8‘-hydroxylation of ABA, which leads to the isomerization of the intermediate 8‘-OH-ABA to phaseic acid (PA) and the subsequent degradation via conversion to dihydrophaseic acid (Fig. 6A ¹⁷ ). Four P450-type monooxygenases CYP707A were identified to catalyze the initial step in A. thaliana. ¹⁵⁵ All of them were present in the same CLOG together with four co-orthologues of tomato (Supplementary Tables 4 and 11; Solyc08g005610, Solyc08g075320, Solyc04g078900, and Solyc01g108210). The expression patterns showed spatial and temporal differences, indicating that individual members might have different importance during growth and development (Fig. 6A; Supplementary Table 18). The second pathway leads to the formation of glycose ester conjugates (ABA-GE) by the activity of several glycosyltransferases (UGT71Bs ¹⁷ ). ABA-GE is considered as storage or transport form of ABA, which accumulates in vacuoles and the apoplast. Translocation to the ER and rapid reactivation of ABA by cleaving the glycose ester conjugates by β-glucosidases (BG1 and BG2) is assumed to occur in response to dehydration. The five tomato co-orthologues to UGT71B and the one to BG1 were found to be expressed in most tissues. BG2, which was assumed to catalyze the conversion of vacuolar stored ABA conjugates, was expressed at very low level (Fig. 6A; Supplementary Table 18).

Transport of ABA Needs Functional Assignment within Orthologous Groups

Several transporters encoded by distinct transporter gene families are involved in the transport of ABA or ABA conjugates across the various membranes (Fig. 6B). ¹⁵⁶ The members of the subfamily G of the large ATP-binding cassette (ABC) protein family are one group of these transporters (ABCG). ¹⁵⁷ In A. thaliana, ABCG25 was the first ABA efflux carrier identified in the plasma membrane. It is expressed mainly in vascular tissues.^158,159 ABCG22 and ABCG40 reside in guard cells and contribute to enhanced import of ABA into stomatal cells. ¹⁶⁰ The CLOGs of ABCG22 and ABCG40 contained five additional co-orthologues to Arabidopsis ABCG22/25 and 14 co-orthologues to ABCG40, for which the functional assignment remains to be elaborated. In total 31 ABCG co-orthologues have been detected in tomato (Supplementary Table 18).

The ABA-importing transporter 1 (AIT1), originally characterized as nitrate transporter (NRT1.2), is involved in cellular import of ABA in the vascular tissues of inflorescence stems, leaves, and roots. ⁷ In the genome of tomato, four co-orthologues to AIT1 were identified (Supplementary Table 18). DTX50 is an ABC transporter of the detoxification efflux carriers/multidrug and toxic compound extrusion (DTX/MATE) family shown to function in efficient ABA export after drought stress. ¹⁶¹ DTX50 plays an important role in the removal of excess ABA levels to prevent hyperaccumulation of the hormone. ¹⁵⁷ Again, four co-orthologues were detected in the genome of tomato (Supplementary Table 18). Finally, two members of the ABCC subfamily in A. thaliana, AtABCC1/MRT1 and AtABCC2/MRT2 (multidrug resistance–associated protein), have been shown to transport ABA-GE across the tonoplast into the vacuole. Both belong to the same CLOG in which 14 tomato genes were also clustered.

Three ABA-specific receptor types associated with distinct cellular localizations have been described (Fig. 6C).^17,48,162 One type is localized to the ER, Golgi, and peripheral plasma membranes and consists of the G protein coupled receptor proteins GTG1, GTG2, and GCR2. Interaction with the G-alpha subunit GPA1 regulates the binding of ABA. Signal transduction from these receptors mainly targets the regulation of membrane channels involved in Ca²⁺ transport and control of water permeability, eg, in guard cells. We identified three tomato co-orthologues of GCR2, and one for GTG1 and GTG2. Interestingly, the latter was only expressed in flower and fruit tissues (Supplementary Table 18).

The subunit H of the Mg²⁺ chelatase complex (CCH) localized in plastids was proposed as an additional ABA receptor. ¹⁶³ The tomato orthologue of CCH was found to be highly expressed in all tissues (Fig. 6C; Supplementary Table 18). Binding of ABA enhances the interaction of subunit H on the cytosolic side with a group of transcription factors from the WRKY family, thereby preventing their translocation to the nucleus. In the absence of ABA, the transcription factors act as negative regulators of ABA response genes.

The third class of ABA receptors consists of soluble proteins seen as entrance components of the “core ABA signaling pathway” (Fig. 6C ⁴⁸ ). These receptors belong to a gene family (14 genes in A. thaliana) known as pyrobactin resistant (PYR), PYR-like (PYL), or regulatory component of ABA receptor (RCAR) which was represented in our study by eight co-orthologues in S. lycopersicum (Supplementary Table 4). The proteins control the activity of downstream components of the signaling pathway by the interaction with protein phosphatase 2C (PP2C), which acts as a negative regulator of ABA response. This inhibition is maintained by the interaction with the active ABI1, 2 enzymes containing the PP2C domain (conserved in all 13 species, Supplementary Table 11). Further, SNF1-related kinases (SnRKs; seven co-orthologues in tomato) regulate the ABA signaling in the absence of ABA or at low concentrations of ABA. This interaction prevents the phosphorylation and activation of numerous transcription factors, ion channels, and other mediators of ABA responses. Alternating binding of PYR/PYL/RCAR to ABI1, 2 controls release and activity of the SnRKs and thereby the activation of a transcription factor cascade cooperating in the regulation of numerous ABA response genes. ¹⁶⁴

Differences in the Localization of Biosynthesis Enzymes from JA in Tomato

Jasmonates are lipid-derived compounds synthesized from α-linolenic acid released from plastid membranes by one of the seven different branches of the lipoxygenase (LOX) pathway. ¹⁶⁵ Subsequently, β-oxidation occurs in peroxisomes and the final step of JA synthesis takes place in the cytoplasm. ¹⁶⁶ Defective in anther dehiscense 1 (DAD1), phos-pholipase1 (PLA1), and phospholipase 2 (PLA2) are involved in α-linolenic acid production. ⁶¹ In A. thaliana, dad1 mutant results in a male sterile phenotype, ¹⁶⁷ indicating that DAD1 is responsible for JA biosynthesis in flowers, while in leaves JA is synthesized by the DGL (Dongle) gene.^168,169 Both enzyme CLOGs contained no co-orthologues from C. reinhardtii and P. patens; thus, it is likely that they evolved in the ancestor of monocots and eudicots (Supplementary Table 5). We identified two co-orthologues for DGL and one orthologue for DAD1 in tomato, but none of them were expressed in the tissues and developmental stages of fruits analyzed (Fig. 7A; Supplementary Table 19). This suggests that another pathway for linolenic acid in tomato may exist or that the pathway is only activated under stress conditions.

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Figure 7

Pathways of jasmonic acid synthesis and signaling.

α-linolenic acid (18:3) acts as a fatty acid substrate for synthesis of 13(S)-hydroperoxylinolenic acid by LOXs. ¹⁷⁰ Except in C. reinhardtii, all plant species contained more than six co-orthologues, with a maximum of 43 co-orthologues in G. max. In total, 18 co-orthologues were identified in tomato; however, a chloroplast targeting signal was only predicted for three of them (Solyc05g014790, Solyc03g122340, and Solyc01g006560), which most likely indicated their involvement in jasmonate synthesis (Fig. 7A; Supplementary Table 12). At least one of them was expressed in each tissue and Solyc01 g006560 was highly expressed in leaves. Allene oxide synthase (AOS; six co-orthologues in tomato) ¹⁷¹ catalyzes the dehydration of 13(S)-hydroperoxylinolenic acid to 12,13(S)-epoxylinolenic acid, which is converted to cis(+)-12-oxophytodienoic acid (OPDA) likely by allene oxide cyclase (AOC). Interestingly, we detected only two co-orthologues of AOS containing a chloroplast targeting signal (Solyc04g079730 and Solyc11g069800) in tomato. At least one of them was expressed in all tissues, similar to the unique tomato AOC orthologue (Fig. 7A). In A. thaliana, transcription of AOC is induced within two hours after wounding and occurs in anthers and pollen grains, ¹⁷² while we observed moderate expression of the tomato orthologue in all tissues (Supplementary Table 19^173,174).

OPDA is transported from plastids to peroxisomes by the action of CTS, ¹⁷⁵ and the expression of the tomato orthologue was at least low or moderate in all tissues (Fig. 7A; Supplementary Table 19). The OPDA reductase (OPR3) catalyzes the reduction of OPDA to 3-oxo-2-(2'(Z)-pentenyl)-cyclopentane-1-octanoic acid (OPC ¹⁷⁶ ) and the orthologue in tomato showed high variance in expression from low to high levels in the tissues analyzed (Fig. 7A). OPDA-CoA intermediate is formed in peroxisomes by 4-coumarate:CoA ligase like (4-CL-like) enzymes such as OPCL1, whose orthologue in tomato is globally expressed (Fig. 7A). ¹⁷⁷ The subsequent β-oxidation to synthesize JA occurs in three cycles ¹⁷⁸ by the action of an acyl-CoA oxidase (ACX1), a multifunctional protein (MFP), and an L-3-ketoacyl CoA thiolase (KAT). ⁶⁰ While two peroxisome targeted co-orthologues of ACX1 and six of MFP were identified in tomato, we could not predict a peroxisomal targeting signal in one of the two co-orthologues of KAT (Fig. 7A; Supplementary Table 12). Interestingly, MFP2 and AIM1 were located in one orthologous group in A. thaliana while six co-orthologues were found in tomato of which the peroxisomal-located enzymes Solyc12g007170, Solyc12g099440, Solyc07g019670, and Solyc01g066620 were at least low expressed in all tissues except Solyc01g066620 (Supplementary Table 19).

JA is subsequently secreted into the cytoplasm, where it can be further utilized either by S-adenosyl-L-methionine:jasmonic acid carboxyl methyltransferase (JMT) to form the volatile JA methyl ester (methyl (+)-7-isojasmonate) or conjugated to isoleucine by the activity of JAR1, which converts JA into the biologically active jasmonyl-iso-leucine.^4,179,180 JMT was only present in eudicots and represented by a single orthologue in tomato, which exhibited a low expression in root, stem, and leaf. In tomato, (+)-7-iso-JA-Ile is exclusively synthesized upon wounding, and in SlJAR1-RNAi lines, JA-Ile is downregulated by 50%-75%, confirming the importance of JAR1 for the conversion of JA to JA-Ile. ¹⁸¹ Remarkably, we observed that the CLOG of JAR1 contained 13 tomato co-orthologues, and one of them (Solyc01g095580) was highly expressed in all tissues except of flowers and stems. Interestingly, the co-orthologue Solyc10g011660showed a high expression in flowers and stems, and by this, we could compensate Solyc01g095580 in both tissues (Fig. 7A; Supplementary Tables 5, 12, and 19). Thus, it needs to be experimentally proven whether Solyc01g095580 is the essential JAR1 orthologue in jasmonate signaling and whether rapid induction of the gene is caused by stress induction during the harvesting procedure.

Jasmonate Response Co-Orthologues show Differences between tomato and Arabidopsis

The main components of JA response are transcription factors of the JA-ZIM-domain (JAZ) repressor family, calcium-related signaling molecules, and JA-related transcription activators. Ca²⁺-dependent phosphorylation and MAPK cascades are also involved in the regulation of JA biosynthesis. ⁶¹ Among the targets of JA signaling are JA biosynthesis genes that form a positive feedback system. ¹⁸²

We observed that the regulation of transcriptional changes triggered by JA synthesis was conserved in eudicots and monocots (Fig. 7B; Supplementary Tables 5 and 12).^183–185 The primary regulators are MYC transcription factors and JAZ repressors are associated in corepressor complexes with NINJA and TPL (Topless; Fig. 7B). None of the eight JAZ repressors in A. thaliana were represented in green algae and moss, and JAZ5, 6, and 9 occurred only in eudicots or were found specifically in A. thaliana (Supplementary Table 5). Degradation of JAZ repressors is mediated by binding of JA-Ile to the F-box protein coronatine-insensitive 1 (COI1) bound to the SCF^Col1 E3 ubiquitin ligase complex (Fig. 7B), which in turn allows the expression of early JA response genes.^186–188 JAZ proteins also bind to MYB21 and MYB24, two transcription factors encoded by genes strongly induced by JA in flower tissues that control stamen development and pollen maturation in A. thaliana.^189,190 For tomato, only three JAZ co-orthologues were identified in two distinct CLOGs, showing the same domain architecture containing a TIFY (transcription factor domain) and a CCT_2 (short plant protein motif) domain (Supplementary Table 12). For two co-orthologues (Solyc07g042170 and Solyc03g118540), high expression levels were found in roots and Solyc07g042170 was at least moderately expressed in all other tissues (Supplementary Table 19).

Impairment of JA signaling leads to severe phenotypes like male sterility in A. thaliana, while in the tomato mutant, jai1-1 female sterility was observed. ¹⁹¹ In line with the essential role of JAI1, the tomato orthologue was expressed in all tissues. Interestingly, expression of the COI1 orthologue was not observed in tomato flowers (Supplementary Table 19), ¹⁶⁵ although it is interacting with JAI1.

Low-expressed Enzymes in Tomato influence GA Biosynthetic Pathway

GAs derive from the diterpene geranylgeranyl diphosphate (GGDP) synthesized in plastids from the isoprenoid precursor isopentenyl pyrophosphate, which is provided mainly by the plastid-resident MEP pathway, but also by the cytosolic MVA pathway (Fig. 8A). ¹⁹² Our analysis showed that both pathways were conserved in mosses, monocots, and eudicots, but the MVA pathway was missing in the green algae C. reinhardtii.^193,194 Remarkably, the co-orthologues from the MEP and MVA pathway showed moderate or high expression in all tomato tissues, whereas the expression of co-orthologues catalyzing the subsequent steps of GA biosynthesis was low in tomato or even not present in some tissues (Fig. 8A, Supplementary Table 20). The chloroplast-localized ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS), which are involved in formation of ent-kaurene, were detected in all plants except in the green algae (Fig. 8A, Supplementary Table 6). Interestingly, expression of tomato CPS and KS co-orthologues was detected if at all at low levels only (Fig. 8A, Supplementary Table 20). GA1 (CPS) and GA2 (KS) in A. thaliana were discriminated on the basis of their domain architecture, which could be confirmed by our orthologue search due to the fact that they fall into two different CLOGs. Inspection of the domain architecture of co-orthologues within the two CLOGs indicated that orthologue search and functional domain architecture might differ, but it could also exemplify an interesting case of domain stealing. ¹⁰⁰ For instance, some of the monocot co-orthologues in the CPS group contain a Prenyltrans_1 domain. This domain is absent in the CPS of A. thaliana, but present in the KS orthologue (Supplementary Table 13).

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Figure 8

Pathways of gibberellin synthesis and signaling. (A) Enzymes and intermediate products of the plastidial and cytosolic pathways for isopentenyl pyrophosphate production unify to the plastidal production of ent-kauren as precursor for gibberellin synthesis (GA₁, GA₄, and GA₃) in the cytosol. GA2ox and GAMT mark the biological inactivation of gibberellin. For further details, see text. The arrows are colored according to the species in which the enzymes were found (Fig. 1A). Expression of the identified genes in tomato is shown as explained in Figure 2 (B) The components involved in gibberellin signaling are presented as interaction scheme. Enzyme activities not expressed by any orthologue gene in tomato are indicated in gray.

Conversion of ent-kaurene to GA₁₂ requires the transport of ent-kaurene into the cytoplasm and several oxidation steps. The first step is catalyzed by the ent-kaurene oxidase (KO), localized in the outer envelope of plastids, which is involved in the simultaneous transfer of ent-kaurenoic acid to the ER. ¹⁹⁵ Subsequent oxidization requires the ER-associated ent-kaurenoic acid oxidase (KAO) for conversion to GA₁₂. However, the nature of the enzyme activity involved in the formation of GA₅₃ by hydroxylation on C-13 is still not clear. The subsequent hydroxylation on C-20 of GA₁₂ or GA₅₃ is catalyzed by cytoplasmic GA20 oxoglutarate-dependent dioxygenases (GA20ox). The activity of other oxoglutarate-dependent dioxygenases like the GA3ox further leads to the production of bioactive GA compounds such as GA₁, GA₄, or GA₃. Deactivation of GAs by C-2 hydroxylation is catalyzed by GA2 oxidases (GA2ox). As in the case of KS, KAO1 and KAO2, co-orthologues of GA3ox and GA2ox were not found in P. patens and C. reinhardtii (Supplementary Table 6), while their expression was low in most of the analyzed tissues (Supplementary Table 20).

Oxidization and conversion of GAs into their epoxides by cytochrome P450 monooxygenases as well as formation of GA methyl esters by the activity of GA methyl transferases (GAMTs) are discussed as other mechanisms of GA deactivation. ⁶⁶ However, these mechanisms appear to be species-specific as co-orthologues to the A. thaliana GAMT were not identified by our approach in any of the selected species except for V. vinifera (Supplementary Table 6).

GA Perception and Signaling is Absent in Green Algae and Mosses

Current models for GA perception and signaling consider three key components. ¹⁹⁶ The first is the soluble, nuclear-localized GA receptor GID1, ¹⁹⁷ which we identified in monocots and eudicots (Supplementary Table 6). The second are the growth repressing DELLA proteins GAI, RGA, and RGL1, 2, and 3 identified in A. thaliana ¹⁹⁸ as well as SLR1 found in rice, ¹⁹⁹ which form a subfamily of the plant-specific GRAS transcription regulators. All of these proteins were grouped in one CLOG also containing co-orthologues from the moss in addition to monocots and eudicots. However, in contrast to A. thaliana that has five GRAS enzymes, many eudicots and monocots contained only one orthologue. The third are the F-box proteins SLY1 and SLY2, ²⁰⁰ which are called GID2 in rice (Fig. 8B ²⁰¹ ). No co-orthologues of these factors were detected in the moss and the green algae. By this, the GA signaling pathway seems to have emerged after mosses. However, co-orthologues of SLY were not found in V. vinifera (Supplementary Table 6). Remarkably, SLY2 co-orthologues were not expressed in the analyzed tomato tissues under normal conditions (Supplementary Table 20).

In the absence of GA, DELLA proteins act as central repressors of GA response by suppressing the activity of PIF transcription factors (PIF3, 4), which are involved in the regulation of growth-promoting GA-responsive genes.^202,203 Binding of GA to the receptor GID1 triggers its interaction with DELLA proteins and causes their release from repression complexes. ²⁰⁴ In turn, simultaneous binding of SLY1/GID2 recruits DELLA proteins for ubiquitination by the SLY1/GID2-dependent E3-ligase complex and subsequent degradation by the proteasome. ²⁰⁴ Furthermore, the level of DELLA proteins is also controlled by the activity of the phytochrome-interacting factor PIF1/PIL5, and the regulation of DELLA activity is done by proteins SCL3 and SPY1.^67,205 In general, PIFs have been evolved specifically in different species, as PIF6 was present only in A. thaliana and we observed PIF1 co-orthologues only in some eudicots (S. tuberosum, S. lycopersicum, M. truncatula, and G. max; Supplementary Tables 6 and 13). Further, our analysis for expression in tomato revealed that most of the enzymes were expressed at low levels under normal conditions (Supplementary Table 20).

BR Synthesis is Conserved in Viridiplantae until Synthesis of Campesterol

As for all other phytosteriods, the biosynthesis of BR derives from isopentenyl pyrophosphate (isopentenyl PP; Fig. 9A), which is conjugated to the triterpene squalene (Fig. 9A).^16,206,207 By the action of CAS1, SMT1, CYP51, FACKEL, HYD1, DWF7, DWF5, and DWF1, campesterol is synthesized, ⁷³ which is the direct C28 precursor of the BR specific pathway. In line with the fact that campesterol is not exclusively a precursor for BR, the pathway was conserved in all analyzed plants with the exception of FACKEL and DWF5 (Fig. 9A, Supplementary Tables 7 and 14). The enzyme FACKEL was only present in Z. mays, S. tuberosum, and A. thaliana, whereas DWF5 co-orthologues were only missing in C. reinhardtii. In tomato, CAS1 co-orthologues showed a moderate expression at maximum and could potentially limit the rate of campesterol synthesis. In contrast, SMT1, CYP51, and DWF5 showed the tendency to be highly expressed in most of the tissues (Supplementary Table 21). Furthermore, HYD1 orthologue was not expressed in flower tissue.

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Figure 9

Pathways of brassinosteroide synthesis and signaling.

The subsequent processing of BR synthesis starting from campesterol as a basic precursor is not yet fully described and the exact order of enzymatic reactions in this part of the BR pathway is still under debate. For our analysis, we depicted the two pathways described by Zhao and Li, ⁷⁶ which are branching during the action of DET2, DWF4 (annotated as CYP724B2 and CYP90B3 in tomato, respectively), and CPD (annotated as CYP90A1 in A. thaliana). Remarkably, only DET2 co-orthologues were found in the green algae and the moss included in our analysis (Supplementary Table 14). For eudicots, the reaction catalyzed by the P450 monooxygenase DWF4 has been proposed as a rate-limiting step in BR synthesis, ²⁰⁸ and transcriptome profiling of tomato co-orthologues revealed only low expression in all tissues for DWF4. In contrast, CPD showed moderate or high expression in all tissues (Supplementary Table 21). For co-orthologues in both routes, mostly no or only low expression was observed in flower tissue. The two routes lead to the production of 6-deoxotyphasterol and the only two additional enzymes identified in A. thaliana are ROT3 (CYP90C1) and CYP90D1, but it is unknown yet whether both enzymes indeed participate in both pathways.

The conversion of 6-deoxotyphasterol to 6-deoxocathaster-one is catalyzed by DDWF1, which is only described in pea, ²⁰⁹ while the corresponding gene in A. thaliana has not been identified yet. Finally, oxidation on the C-6 by BR6ox leads to CS, the immediate precursor of BL. Br6ox (CYP85A1 and CYP85A2 in A. thaliana) has been originally identified as an extreme dwarf (d^x) allele of the tomato DWARF locus (D). CYP85A2 (Br6ox2), but not CYP85A1 (Br6ox1), drives the conversion of CS to BL. Expression analysis of tomato co-orthologues in different tissues showed that only one co-orthologue was expressed in both cases. For the co-orthologues of Br6ox, only moderate expression was detected in fruit ripening stages, whereas for ROT3/ CYP90D1, moderate expression was observed in root, stem, and leaf (Supplementary Table 21).

Alternative Modes of BR Signaling in Monocots and Moss

Because long-distance transport is not shown for BR or derivatives so far, it is widely assumed that they act in close vicinity to the places of synthesis. ²¹⁰ Binding of BRs to the extracellular leucine-rich repeat domain of the serine/threonine receptor kinase BRI1 initiates the release of the inhibitor protein BKI1 and formation of heterodimers with BAK1 (Fig. 9B^211,212). While we detected co-orthologues of BAK1 and BRI1 in all plants except the green algae C. reinhardtii, the inhibitor BKI1 was identified only in eudicots, which suggests an alternative mode of regulation in monocots (Supplementary Table 7). Interestingly, the CLOG of BAK1 contained seven to 30 co-orthologues per species in eudicots and monocots. However, the identified co-orthologues showed a large heterogeneity in their domain architecture (Supplementary Table 14).

Autophosphorylation and sequential transphosphorylation of the cytoplasmic kinase domains fully activate the receptor complex and triggers the downstream signal transduction by activating BSK1, which in turn phosphorylates BSU1. Interestingly, BSK1 was low expressed during fruit ripening and in the mature fruit (Supplementary Table 21). BSU1 acts as a protein phosphatase and inactivates the serine/threonine kinase BIN2 by dephosphorylation. In the absence of BR, BIN2 is constitutively active and controls the phosphorylation of BZR1 and BES1/BZR2, two transcription factors involved in the regulation of BR-responsive genes. We observed that this regulatory network was mainly conserved in all plants except for green algae. Both transcription factors are kept inactive by either rapid proteasomal degradation or cytoplasmic retention through interaction with 14-3-3. ⁷⁴ Only BZR1 and BES1/BZR2 were not identified in P. patens, which suggests that species-specific transcription factors might exist in moss (Supplementary Table 7).

Besides controlling the expression of growth-promoting genes, BZR1 and BZR2 also feedback the expression of upstream signaling components and genes involved in BR biosynthesis. Among these, SBI1 mediates the methylation of PP2A, which in turn is translocated to the plasma membrane and promotes the inactivation of the internalized BRI1 receptor by dephosphorylation (Fig. 9B). In contrast to PP2A, SBI1 was not identified in the eudicot S. tuberosum, which could be related to the incomplete genome draft or an analogous redundant function.

Supplementary Tables 1-7

Survey of genes involved in phytohormone biosynthesis, transport, and signaling in A. thaliana and their co-orthologues in selected Viridiplantae. Analyzed phytohormones are auxin (S1), ethylene (S2), cytokinin (S3), abscisic acid (S4), jasmonic acid (S5), gibberellin (S6), and brassinosteroid (S7). The corresponding AGI number, gene, and mutant names, description in database, number of species containing co-orthologues, number of co-orthologues in the indicated plant species and the overall distribution in the groups of eudicots, monocots, mosses, and algae are indicated. The selected plant species are: Atha, A. thaliana; Bdis, B. distachyon; Crei, C. reinhardtii; Gmax, G. max; Mtru, M. truncatula; Osat, O. sativa; Ppat, P. patens; Ptri, P. trichocarpa; Sbic, S. bicolor; Slyc, S. lycopersicum; Stub, S. tuberosum; Vvin, V. vinifera; Zmay, Z. mays.

Supplementary Tables 8-14

Survey of co-orthologues involved in phytohormone biosynthesis, transport, and signaling in the analyzed plant genomes with prediction of intracellular localization and domain structure. Analyzed phytohormones are auxin (S8), ethylene (S9), cytokinin (S10), abscisic acid (S11), jasmonic acid (S12), gibberellin (S13), and brassinosteroid (S14). The selected plant species are: Atha, A. thaliana; Bdis, B. distachyon; Crei, C. reinhardtii; Gmax, G. max; Mtru, M. truncatula; Osat, O. sativa; Ppat, P. patens; Ptri, P. trichocarpa; Sbic, S. bicolor; Slyc, S. lycopersicum; Stub, S. tuberosum; Vvin, V. vinifera; Zmay, Z. mays.

Supplementary Tables 15-21

Expression of tomato co-orthologues involved in phytohormone biosynthesis, transport, and signaling in different tissues: root, stem, leaf, flower, and fruits at the three developmental stages, namely, mature green (fruit_mature), breaker stage (berry_breaker), and 10 days after turning red (berry_10 days). Analyzed phytohormones are auxin (S15), ethylene (S16), cytokinin (S17), abscisic acid (S18), jasmonic acid (S19), gibberellin (S20), and brassinosteroid (S21).