Genome-Wide Analysis of the Polyamine Oxidase Gene Family In

bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.189209; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Genome-wide analysis of the polyamine oxidase gene family in

wheat (Triticum aestivum L.) reveals involvement in temperature

stress response

Authors:

Fatemeh Gholizadeh, Ghader Mirzaghaderi*

Address:

Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Kurdistan,

P. O. Box 416, Sanandaj, Iran

*Corresponding author, E-mail: [email protected], ORCID: 0000-0002-4578-3374

Phone: +98-8733620552

Fax: +98-8733620553

Short title:

PAO gene family in common wheat

Abstract Amine oxidases (AOs) including copper containing amine oxidases (CuAOs) and

FAD-dependent polyamine oxidases (PAOs) are associated with polyamine catabolism in the

peroxisome, apoplast and cytoplasm and play an essential role in growth and developmental

processes and response to biotic and abiotic stresses. Here, we identified PAO genes in

common wheat (Triticum aestivum), T. urartu and Aegilops tauschii and reported the genome

organization, evolutionary features and expression profiles of the wheat PAO genes (TaPAO).

Expression analysis using publicly available RNASeq data showed that TaPAO genes are

expressed redundantly in various tissues and developmental stages. A large percentage of

TaPAOs respond significantly to abiotic stresses, especially temperature (i.e. heat and cold

stress). Some TaPAOs were also involved in response to other stresses such as powdery

mildew, stripe rust and Fusarium infection. Overall, TaPAOs may have various functions in

stress tolerances responses, and play vital roles in different tissues and developmental stages.

Our results provided a reference for further functional investigation of TaPAO proteins.

Keywords: polyamine oxidase (PAO), polyamine, biotic and abiotic stress, wheat

Introduction

Common wheat (Triticum aestivum L., 2n = 6x = 42; AABBDD genome), is one of the

most important cereal crops. It is constantly exposed to abiotic and biotic stresses such as heat,

cold, salinity, drought and various fungal diseases. These stresses reduce growth and yield and

may cause plant death. Therefore it is essential to understand how wheat adapts and survives

in stressful environments, and to develop methods to increase its tolerance under environmental

stresses [1].

Polyamines (PAs), are small aliphatic amines of low molecular weight that are involved in

various developmental processes in living organisms. Main PAs in cells include diamine

putrescine (Put), triamine spermidine (Spd), tetramines spermine (Spm), cadaverine (Cad) and

thermospermine (T-Spm). Due to their cationic nature, polyamines are capable of binding to

negatively charged molecules such as RNA and DNA and affect gene expression, protein

synthesis and regulation of ion channels [2]. De novo production of PAs in plants includes Put

production directly from ornithine by ornithine decarboxylase (ODC), or indirectly from

arginine by arginine decarboxylase (ADC) [1]. Put is then converted into Spd by spermidine

synthase with the addition of an amino propyl moiety donated by decarboxylated S-adenosyl

methionine (dcSAM). Similarly, Spm (and its isomer T-Spm) is formed from Spd via Spm

synthase, with the same amino propyl group rendered by dcSAM [3, 4] (Fig. 1).

PAs can be oxidized by copper‐containing diamine oxidases (CuAOs or DAOs) and flavin-

containing (FAD‐containing) polyamine oxidases (PAOs) [5]. DAOs mainly catalyze the

oxidation of Put and Cad producing 4‐aminobutanal, ammonia (NH3) and hydrogen peroxide

(H2O2) [6, 7]. POAs are divided into two major groups. The first group catalyzes Spd and Spm

to produce 1,3‐diaminopropane (DAP), H2O2, and N‐(3‐aminopropyl)‐4‐aminobutanal or 4‐

aminobutanal, which is referred to as the terminal catabolism (TC) pathway [5, 7, 8]. The

second group is involved in the back conversion (BC) pathway by converting Spm back to Spd

and Spd to Put [7, 9].

Plants accumulate osmolyte compounds in response to abiotic stresses such as drought and

salinity. Major cellular osmolytes including proline, glycine betaine, and PAs are found in

plants, animals, and bacteria [10]. In plants, PAs are essential for development and stress

response. Many plant processes such as embryogenesis, organogenesis, particularly flower

initiation and development, fruit setting and ripening, as well as leaf senescence, require PAs

[3, 11]. Cells need to maintain the homeostasis of PAs through their modulation, biosynthesis,

conjugation, and transport, since high concentrations of polyamines are highly toxic [12].

Spd and Spm and Put levels are differentially regulated by environmental stresses [13],

although the mechanism of PA action in response to stresses still remain unclear. Put levels are

increased with low potassium (K+) availability in plants suggest that Put and its catabolites

possess a potential in controlling cellular K+ and Ca2+ [14]. During drought, the PA pathway is

activated which leads to a Put to Spd canalization that is ABA-dependent. Drought tolerant and

sensitive cultivars seem to be different in their capacity to accumulate different PAs over a

minimum threshold [15].

H2O2 produced through PA oxidation is involved in a hyper-sensitive (HR) reaction that

can lead to bacterial pathogen tolerance [16]. Exogenous Spm results in HR-mediated

resistance of Arabidopsis leaves to cucumber mosaic virus via the induction of the expression

of some H2O2 -dependent signaling components and transcription factors. Addition of a PAO

inhibitor represses the activation of defense genes and alleviates ROS generation and HR,

confirming that PAO is involved in the resistance response [17]. There is evidence that PA

oxidation in the apoplast together with the generated reactive oxygen species (ROS) are

involved in programmed cell death (PCD) and xylem differentiation [3]. The transcript levels

of PA synthesis genes, and the activities of corresponding enzymes are responsive to stresses,

providing a relationship between polyamine and stresses [1]. Plant PAOs play significant roles

in metal (e.g. aluminum, copper, and cadmium) toxicity tolerance [18-22]. In wheat, the cell

wall-bound PAO (CW-PAO) oxidized Spd and generated H2O2 under aluminum toxicity but

Put application resulted in plant tolerance against Aluminum-induced oxidative stress via

inhibiting PAO activity and hence lowering H2O2 production [20].

PAO genes have been isolated and characterized from several model plants. One of the

first polyamine oxidases identified was a FAD-based PAO in maize apoplast, a 53-kDa 4

monomeric glycoprotein enzyme [23]. Most of the identified plant PAO genes such as A.

thaliana AtPAO1 to AtPAO5 are involved in the BC pathway. AtPAO1 and AtPAO5 are

located in the cytoplasm, while AtPAO2, AtPAO3 and AtPAO4 have a peroxisomal

localization [24-26]. AtPAO1 is involved in biotic and abiotic stress tolerance and may play

roles in root development and fertility. On the other hand AtPAO2 might be involved in root,

shoot, leaf, and flower development. AtPAO3 and AtPAO4 are expressed in all tissues and

whole growth stages and show similar expression patterns [27, 28]. Rice harbors seven PAO

genes. OsPAO3 and OsPAO5 are very similar and highly expressed in both the seedling stage

and in mature plants, while the other PAO members are only expressed at very low levels in

all plant tissues. OsPAO4 and OsPAO5 prefer to use Spm and T-Spm as substrates, but cannot

oxidize Spd to Put. Therefore, OsPAO3 catalyzes a full BC-type pathway, while OsPAO4 and

OsPAO5 only catalyze a partial BC-type pathway [4].

In the present study, polyamine oxidase genes were identified in T. aestivum, T. urartu and

Aegilops tauschii using bioinformatic approaches and their gene structure, conserved protein

motifs and domains and phylogenetic relationships were analyzed. Furthermore, we examined

the expression of the wheat PAO genes over different tissues and developmental stages and in

response to biotic and abiotic stresses.

Materials and Methods

Identification of PAO genes

Polyamine oxidase genes of common wheat (T. aestivum) and its relatives T. urartu and

Ae. tauschii, were identified by BLASTP search, Hidden Markov Model (HMM) analysis and

validation of conservative domains. For this, the Arabidopsis and rice PAO protein sequences

(supplementary File 1) were used as queries to perform BLASTP searches against the T.

aestivum, T. urartu and Ae. tauschii genome (E-value < 1e-5) in the EnsemblPlants database 5

at https://plants.ensembl.org. Furthermore, an HMM matrix of five AtPAO and seven OsPAO

protein sequences was used to search the PAO proteins in jackhmmer

(https://www.ebi.ac.uk/tools/hmmer/search/jackhmmer) [29]. We then selected the unique

sequences of the above two search results and checked them for the presence of each of the

amine oxidase domains (Pfam: PF01593) alone or in combination with copper amine oxidase

(N2 and/or N3-terminal), using the Pfam (https://pfam.xfam.org) and InterPro

(http://www.ebi.ac.uk/interpro) databases. Proteins with amine oxidase in combination with

other extra domains were excluded, as such architectures are known to have functions different

from PAO. For example, plant lysine histone demethylases which possess an additional

SWIRM domain are involved in demethylation of mono- and di-methylated lysines of histones

[30]. Other described genes such as zeta-carotene desaturase, protoporphyrinogen oxidase,

prolycopene isomerase and protein FLOWERING locus D-like protein were also excluded.

Identification of orthologs and homoeologs

PAO homoeologous genes and pairwise gene orthologs among T. aestivum, T. urartu, Ae.

tauschii, A. thaliana and Oryza sativa were identified through the “homoeologous” and

“orthologoues” links in the gene-based display of the EnsemblPlants summary page for each

target gene. PAO genes were mapped to their respective locus in the wheat genome in a circular

diagram using shinyCircos [31] where homoeologous chromosomes were aligned close

together and banded according to the general FISH patterns of pTa535-1 and (GAA)10 probes.

Characterization of TaPAO genes

Characteristics of each of the identified amino oxidase proteins such as isoelectric point

(pI), amino acid sequence length (AA) and molecular weight (MW) were obtained from the

ProtParam website at https://web.expasy.org/protparam [32]. A GFF3 annotation file

containing the locations of TaPAOs in genome and their structural information was extracted

from the wheat GFF3 file and the exon-intron structures was displayed using the Gene 6

Structure Display Server (GSDS, http://gsds.cbi.pku.edu.cn) [33]. The conserved domains of

the TaPAO protein sequences were searched from Pfam [34] and MEME [35] websites and the

resulting files were visualized in TBtools software [36]. Wheat and rice PAO protein sequences

were also aligned in Jalview [37] and the locations of the domains identified by MEME, were

determined on the alignment output file.

Phylogenetic analysis

Multiple sequence alignment of the full-length protein sequences of the identified PAO

proteins was performed using the “msa” package [38] of R version 3.6.1 (The R Project for

Statistical Computing, Vienna, Austria). Subsequently, a neighbor-joining tree was obtained

with 100 bootstrap replicates using the “ape” package [39] and used to generate a tree in R

using the “ggtree” package [40].

Expression analysis of TaPAO genes using RNAseq

RNAseq data of 30 TaPAO genes was retrieved from www.wheat-expression.com [41] as

processed expression values in transcripts per million (TPM) for all the available tissues and

developmental stages [42] and for response to different stresses including Fusarium [43, 44],

cold [45], Zymoseptoria [46], heat and drought [47], phosphorous starvation [48], powdery

mildew [49] and PEG (https://www.ebi.ac.uk/ena/browser/view/PRJNA306536). TaPAO gene

expression values were transformed and used to generate barplots in R. Count matrix data of

all experiments were also downloaded and used for differential gene expression analysis, using

the DESeq2 package [50] to statistically compare the mean expression level of each TaPAO

gene between control and stress conditions. A heatmap was generated from log2(TPM+1)

transformed values of TaPAO genes over developmental stages using R package “pheatmap”.

Ternary plots were generated from the stress response data using the R package ggtern [51].

For this, genes with zero expression in all homoeologs were excluded.

Detecting alternative splicing events among TaPAOs

Wheat genome sequences and annotations (IWGSC RefSeq v1.0) [52] were downloaded

from https://plants.ensembl.org/info/website/ftp/index.html. In order to detect and visualize the

alternative splice variants, we firstly downloaded RNAseq reads [SRP043554, 45] from

https://www.ebi.ac.uk. RNAseq data belong to the wheat plants (‘Manitou’ cultivar) in three-

leaf stage at normal (grown at 23°C for 4 weeks after germination) and cold stress (grown at

23°C for 2 weeks followed by 4°C for another 2 weeks) conditions. After removing the low

quality reads and inspecting for adapter sequences, the raw RNA sequence data from each

sample were mapped to the wheat reference genome using HISAT2 and transcripts were

assembled and merged using StringTie with default settings [53]. Normalization of abundance

estimates as FPKM (fragments per kilobase of transcript per million mapped reads) values,

differential gene and transcript expression analysis and graphical displaying of alternative

splice variants were done using the “ballgown” package [54].

Results

Identification of PAO proteins in common wheat, T. urartu and Ae. tauschii

BLASTP and the Hidden Markov Model (HMM) matrix of Arabidopsis and rice

polyamine oxidase genes (Supplementary File 1) was used to search the amino oxidase proteins

in common wheat, Ae. tauschii and T. urartu protein databases. In total, after verification of

the identified sequences for the presence of each amino_oxidase domain (Pfam: PF01593) or

copper amine oxidase-catalytic domain, either alone or in combination with copper amine

oxidase (N2 and/or N3-terminal), 30 PAO genes in T. aestivum, 6 PAO genes in T. urartu and

8 PAO genes in Ae. tauschii were identified. These genes were named TaPAO1 to TaPAO11,

followed by the name of the harbouring chromosome. For those identified PAO genes which

were orthologous to rice PAOs, the same numbers were assigned as for the rice PAO genes

(Table 1). 8

Phylogeny and characterization of PAO genes

The sequence length of TaPAO proteins ranged from 340 (TaPAO2-2A) to 585 (TaPAO8-

1A, TaPAO8-5B and TaPAOUn) amino acids. The average molecular weight was 54.68 kDa,

varying between 37.87 kDa (TaPAO2-2A) and 62.42 kDa (TaPAO8-5B). The isoelectric points

(pI) of TaPAO members ranged from 5.02 (TaPAO2-2A) to 9.30 (TaPAO7-4A), with an

average of 6.11, showing a weak acidity ( Table 1). In order to identify the evolutionary

relationships between PAO members, a phylogenetic tree of 56 PAO protein sequences

belonging to T. aestivum, T. urartu, A. tauschii, O. sativa and A. thaliana was constructed using

protein sequences based on the neighbor-joining method. The tree clustered the PAOs into

seven clades (Fig. 2). Clade I contains four TaPAO11 homoeologs plus AetPAO11-7D of Ae.

tauschii. Clade II was composed of TaPAO9 and TaPAO8 homoeologs, TaPAOUn, and

TaPAO2-2A. clade III was composed of TaPAO1 homoeologs plus AetPAO1-3D of Ae.

tauschii together with OsPAO1 and AtPAO5. Clade IV contained TaPAO4 and five

homoeologs together with their orthologs from T. urartu, Ae. tauschii and O. sativa plus

AtPAO4. Clade-V had eight members including TaPAO3 homoeologs together with their

orthologs from T. urartu, Ae. tauschii and O. sativa plus AtPAO2 and 3. Clade VI contained

only AtPAO1, which appeared significantly different from other characterized PAOs. Clade

VII was the biggest clade with 17 PAO proteins including TAPAO6 and 7 homoeologs together

with their Ae. tauschii, T. urartu orthologs. O. sativa OsPAO2, OsPAO6 and OsPAO7 proteins

are also in the clade VII which are involved in the TC catabolism pathway (Fig. 1). Taken

together, it seems that the identified wheat PAOs in the present study were not equally

distributed among the different clades. Based on the retrieved data from EnsemblPlants,

TaPAO5-2D, TaPAO6-7A, TaPAO7-4A, TaPAO11-7D and all the Ae. tauschii genes produces

multiple splice variant (Table 1).

Analysis of chromosomal locations of TaPAO genes

A physical map of the location of the TaPAO genes on the A, B, and D chromosomes is

illustrated in Fig. 3. The TaPAO genes were mapped to 16 wheat chromosomes plus the

unassembled (Un) part of the genome. Homoeologs were connected using central links.

Homoeologous chromosomes were aligned close together and banded according to the general

FISH patterns of pTa535-1 and (GAA)10 probes. The TaPAO genes showed uneven distribution

across the A, B, and D subgenomes with a higher density on homoeologous group 2, and

absence on chromosomes 1B, 1D and 6A, 6B and 6D. TaPAO3, TaPAO4 and TaPAO5 showed

a similar exon/intron structure (Fig. 4) and were located together on the distal end of the long

arm of homoeologous group 2, with the same order. TaPAO6 and TaPAO11 were also located

close together on homoeologous group 7A, 7B and 7D but did not show noticeable structural

similarity.

Structure, domain and motif analysis of TaPAO genes

Exon–intron structural diversity within a gene family is an important clue for the

evolutionary and functional analyses of gene family members. Gene structure, exons and

introns were obtained for the identified 30 TaPAO genes to interrogate their genomic

organization ( Fig. 4A). Based on the wheat genome annotation, most TaPAO genes have

introns in their structure and the number of exons varied from 1 (TaPAO9-2A, TaPAO9-2B,

TaPAO1-3A and TaPAO1-3B) to 11 (TaPAO5-2B).

Protein domain analysis showed that most TaPAO members contained a typical

amino_oxidase catalytic domain (alone or in combination with DAO) plus an NAD/FAD

binding domain, with only TaPAO4-2A/-2B/-2D lacking an NAD/FAD binding domain (Fig.

4B). The MEME motif search tool identified six conserved motifs in TaPAO proteins (Fig. 5).

The distribution patterns of these motifs in TaPAO proteins is shown in Fig. 4C. Motif 3 is

present in all TaPAO proteins except TaPAO2-2A. Motif 6 uniformly distributed to all 10

TaPAOs except TaPAO11-7A/-7B and TaPAO2-2A. Motif 1 was available in all TaPAO

except TaPAO2-2A, TaPAO1-3A/3B/3D, TaPAO9-2A/2B, TaPAO8-1A/5B/5D and TaPAO-

Un. Motifs 2, 4 and 5 were present in all TaPAOs except TaPAO2-2A, TaPAO9-2A/2B,

TaPAO8-1A/5B/5D and TaPAO-Un (Fig. 4C).

Expression profile analysis of TaPAOs under developmental stages

Analysis of expression profiles of TaPAO genes at various tissue and developmental stages

using the expVIP data revealed that most TaPAOs are differentially expressed during

developmental stages. For example, TaPAO3-2A/2B, TaPAO4-2A/2B/2D and TaPAO5-

2A/2B/2D are highly expressed in specific tissues and developmental stages. The expression

levels of TaPAO11-7D increased dramatically in some tissues such as leaf sheath, ligule, spike

and spikelet during developmental stages. TaPAO8-1A/5B/5D genes also showed a clear tissue

and developmental specific expression pattern and mainly downregulated in shoot, root and

most parts of spike such as flower, ovary, anther, embryo and grain (Fig. 6). On the other hand,

TaPAO9-A/B/C, TaPAO7 and TaPAO10 are less responsive to different conditions, tissues and

developmental stages, although some homoeologs of these genes were active in some tissues

and developmental stages (Fig. 6 and Fig. 7).

Expression profiles of TaPAOs under biotic and abiotic stresses

The differential expression of TaPAOs under biotic stresses (powdery mildew pathogen,

Zymoseptoria tritici, stripe rust and Fusarium graminearum pathogen infections) and abiotic

stresses (cold, heat, drought, heat and drought, phosphorus starvation and PEG) was assessed

using the downloaded RNAseq data from expVIP. Results show that the expression of

TaPAO8, TaPAO3, TaPAO4, TaPAO5, TaPAO1-3A and TaPAOUn was significantly

upregulated in the leaf of the ‘Manitou’ cultivar under cold stress. However, TaPAO11-

7A/7B/7D were downregulated under the same condition ( Fig. 7A). Expression profiles of

TaPAO-7D were also slightly downregulated under phosphorus starvation ( Fig. 7J).

Furthermore, the transcript expressions of TaPAO3, TaPAO4 and TaPAO5 homoeologs were

significantly increased under heat or under a combination of heat and drought stresses relative

to normal condition in seedling leaves of the ‘TAM 107’ cultivar (Fig. 7D and E), but these

genes were not significantly affected by drought stress ( Fig. 7F). An expression pattern

relatively similar to heat stress was observed for TaPAO3, TaPAO4 and TaPAO5 homoeologs

under PEG treatment, although they showed less expression abundance compared to under heat

stress conditions (Fig. 7H and G). Contrary to the cold (A), heat and drought (B) and heat (C)

stresses, the expression of TaPAO11 homoeologs was significantly increased under PEG

treatment, especially in the ‘Giza 168’ cultivar. Interestingly, TaPAO3 and TaPAO4 genes were

differentially expressed between ‘Giza 168’ and ‘Gemmiza 10’: while the transcript levels of

these genes decreased under PEG in ‘Giza 168’, expression of some genes, such as TaPAO4

significantly increased under similar condition in ‘Gemmiza 10’.

Although some other genes nd homoeologs were differentially expressed in other

experiments, high variation in the data prevented reliable conclusions ( Fig. 7K, L). For

example, the expression of TaPAO4 homoeologs was significantly increased in coleoptile

sheath enclosed shoot tissue of common wheat ‘Chara’ three days after inoculation with F.

graminearum ( Fig. 7C). Some TaPAOs were also differentially expressed between non-

inoculated and inoculated leaves of the ‘N9134’ cultivar seven days after stripe rust and

powdery mildew stress treatment (Fig. 7K, L).

Expression changes of TaPAO genes were also shown in ternary plots for the first three

experiments of Fig. 7M, N and O. Ternary plots for the other TaPAO genes are presented in

supplementary File 2, Fig. S1. Wheat ternary plots, provide an immediate view about the

relative expression and abundance of homoeologous genes from each of the wheat three

subgenomes. For example, the position of TaPAO11 on the plot shows that it is dominantly 12

expressed from the D subgenomes (supplementary file 2 Fig. S1, A-I and Fig. 7M), while

TaPAO1 is mainly expressed from the A subgenomes.

Involvement of alternative splicing in TaPAO genes

To explore alternative splicing in TaPAO genes, the RNAseq data (45.31 Gb) from the

leaves of common wheat cultivar ‘Manitou’ exposed to normal (23°C) and cold stress (4°C)

conditions (accession number: SRP043554) was downloaded and aligned to the recent wheat

reference genome. The overall alignment rate was 93.61%. Transcripts were assembled using

StringTie. Differential transcript expression analysis and graphical displaying of alternative

splice variants were done using the “Ballgown” package [54]. Compared to the number of

splice variants mentioned for each gene in EnsemblPlants, novel isoforms were identified for

12 out of 30 TaPAO genes (Table 1). Because the wheat annotation file was used by StringTie

during the assembly, most of the identified transcript should be due to alternative splicing.

Structure and expression levels of distinct isoforms of the TaPAO5-2D gene under normal

(23°C) and stress (4°C) conditions are illustrated in Fig. 8, where isoforms expressed at higher

levels than the others are indicated by the darker color. Structure and expression levels of

isoforms for the other TaPAO genes are presented in Supplementary File 3, Fig. S2. For most

genes, different isoforms responded differently between normal and stress conditions (Fig. 8,

supplementary file 3, Fig. S2). Among the TaPAO genes, we did not identify any isoforms that

were available only in one condition.

Discussion

Structural characterization of polyamine oxidase genes (PAOs) in wheat

In the present study, we identified six PAO genes in diploid T. urartu, eight in diploid Ae.

tauschii and 30 in hexaploid wheat (T. aestivum) by genome-wide approaches. We also

structurally and functionally characterized the TaPAO genes using the publicly available 13

RNAseq data. Previous studies have identified five PAO members in A. thaliana [55], seven

in rice [4], two in barley [56], one in maize [57], seven in tomato [58], six in sweet orange [1],

five in Brachypodium distachyon [59] and twelve in upland cotton [60]. AtPAO24, and

OsPAO35, are believed to localize in peroxisomes based on possessing

(S/A/C)(K/R/H)(L/M), in their C-termini which is a putative type -I peroxisomal targeting

signal called PTS1 [4, 24]. Presence of SRL sequence in the C-termini of wheat TaPAO3 and

TaPAO5 (Fig. 5) suggests that these proteins are localized in peroxisomes of wheat cells.

The identified TaPAO genes are distributed on 16 out of 21 wheat chromosomes plus the

unassembled (Un) chromosome. As seen in the phylogenetic tree, each of the TaPAO

homoeologous members aligned together in the same clade along with their T. urartu and Ae.

tauschii orthologs (Fig. 2). The TaPAO genes generally showed an uneven distribution across

the A, B, and D subgenomes. Similar biased distribution of gene family members is

widespread. For example, TaWD40, TaGST and TabZIP family members are unevenly

distributed across wheat chromosomes [61-63]. A high structural similarity of exon/intron

structure between TaPAO3, TaPAO4 and TaPAO5, and their close affinity at the distal end of

the long arm of homoeologous group 2 suggest that a gene duplication event might be involved

in the evolution of these genes [64].

Expression profile analysis of TaPAOs during developmental stages

Tissue expression profile analysis revealed that many TaPAOs are expressed in a redundant

manner in different tissues during developmental stages in bread wheat (Figure 5), supporting

the idea that PAOs are involved in various tissues during all developmental processes in all

living organisms [2, 6, 65].

Expression profiles analysis of TaPAOs in response to abiotic stress

It is believed that PA molecules and PAOs also participate in responses to various abiotic

stresses [6, 18, 65]. This has been specifically supported by the presence of putative cis-acting

elements in the promoter region of polyamine biosynthetic genes including ADC and SAMDC

which are regulated by transcription factors such as MYB, ABF and WRKY [66-68].

Concordantly, identification of consistently up- and downregulated expression patterns for a

number of TaPAOs such as TaPAO8, TaPAO4, TaPAO5 and TaPAO11 under cold, drought or

heat stresses suggest the involvement of PAO genes in multiple abiotic stress responses (Figure

6). Specifically, TaPAOs clearly responded to low and high temperatures. A similar

temperature response has been suggested for PAO genes of cotton [60]. Similarly, MdPAO2

expression was upregulated in apple fruit by elevating the CO2 concentrations under low-

temperature/low-O2 storage [69]. In tomato, SlPAOs respond to abiotic stresses including heat,

wounding, cold, drought, and salt [58].

In wheat, polyamine oxidases, were salt-induced in a salinity-tolerant genotype and

showed higher expression compared with a salt-treated wild type, indicating that TaPAOs may

play important roles in salinity tolerance as well [70]. TaPAOs have also been involved in

osmotic stress: both abscisic acid pre-treatment and PEG induced osmotic stress, increased the

Put, but decreased the Spm contents in wheat leaves, suggesting a connection between PA

metabolism and abscisic acid signalling that leads to the controlled regulation and maintenance

of Spd and Spm levels under osmotic stress in wheat seedlings [71]. Compared to high

temperature alone, high temperature plus exogenous application of Spm and high temperature

plus Spd significantly increased grain weight of a heat-resistant wheat variety by 19% and 5%,

and of a heat-sensitive variety by 31% and 34%. Spm, Spd, and proline contents also increased

significantly, while Put contents decreased during grain filling indicating that exogenous Spm

and Spd could ameliorate heat damage during grain filling [72]. 15

Expression profile analysis of TaPAOs in response to biotic stress

Only a few TaPAOs significantly responded to biotic stresses during disease development

but this was genotype and stress-type dependent and varied between experiments. This is not

surprising because gene expression in response to biotic stress has been shown to vary

significantly based on environmental conditions. For example, F. graminearum produces a

different gene expression pattern when infecting diverse tissue types or at different stages of

infection in wheat [73]. Differential gene expression patterns could also be dependent on the

specific isolates infecting host genotypes [74].

Experiment SRP060670 (i.e. Fig. 6B) was the only case where TaPAO11 genes which are

located on the long arm of homoeologous group 7, were not expressed under both normal and

Fusarium stress conditions. This result suggests that the examined wheat genotype in this case

might be a ditelocentric addition line CS-7EL(7D) where the 7DL chromosome arm has been

substituted by 7EL arm of Thinipyrum elongatum [43], subsequently affecting gene expression.

Differential response of homoeologous genes

Differential response of homoeologous genes in allopolyploids is common when the plant

is subjected to stresses. Here, unequal expression of homoeologs in response to stress was

observed for some TaPAO genes such as TaPAO11 under high temperature (Fig. 7D, E) and

phorphorus starvation (Fig. 7J). Dong and Adams (2011) investigated the expression patterns

of homoeologs in response to heat, cold, drought and high salt stresses in allotetraploid cotton

(Gossypium hirsutum) and observed variation in the contribution of homoeologous genes to

abiotic stresses [75]. Similarly, some homoeologs of Coffea canephora which are involved in

the mannitol pathway, presented unequal contributions in response to drought, salt and heat

stresses [76]. While PA-related genes play crucial roles in stress response, the mechanisms of

this PA reaction are not clear. Some evidence suggests that PAO enzymes respond to stress

mainly by modulating the homeostasis of reactive oxygen species (ROS) [1], but a clear 16

understanding of the biochemical functions of PAO proteins requires more experimental

investigation.

Involvement of alternative splicing in TaPAO genes

Among the 30 TaPAO genes, 15 produced more than one isoform while only 3 TaPAO genes

had alternative splice variants in EnsemblPlants. In total, 30 alternative splice variants were

identified in wheat cultivar ‘Manitou’. Therefore, a major proportion of TaPAO transcript

diversity is due to alternative splicing. Observation of a large fraction of novel isoforms in

RNAseq data is common. It is believed that about 60% of intron-containing genes are

alternatively spliced in plants [77, 78]. For example, 63% of intron containing genes are

alternatively spliced in soybean, and on average, each AS gene contain six to seven AS events

[78]. In common wheat, 200, 3576 and 4056 genes exhibited significant alternative splice

pattern changes in response to drought, heat, and a combination of heat and drought stresses,

respectively, implying that expression patterns of alternative splice variants are significantly

altered by heat rather that drought [79]. Moreover, if RNAseq data from samples belonging to

different developmental stages and extreme conditions were to be examined, a higher

proportion of alternatively spliced genes and splice variants would likely be identified.

Alternative splicing might also observed in different tissues and developmental stages [80].

But in the present study, all the TaPAO genes were constitutively alternatively spliced in all

samples.

Conclusion

We identified and characterized 30 PAO genes in common wheat that unevenly distributed

across the wheat chromosomes. TaPAO genes were expressed redundantly in various tissues

and developmental stages but a major fraction of TaPAOs responded significantly to abiotic

stresses especially to temperature (i.e. heat and cold stresses). Some TaPAOs were also 17

involved in responses to other stresses such as, powdery mildew, stripe rust and Fusarium

infections in wheat. Overall, TaPAOs likely function in stress tolerances and play vital roles in

different tissues and developmental stages. To understand the exact mechanisms of polyamine

catabolism and biological functions of TaPAOs, more genetic and biochemical experiments are

required. Our results provide a reference for further functional investigation of TaPAOs

proteins.

Acknowledgements

We thank Professor Annaliese Mason (Justus Liebig University, Germany) for providing

helpful corrections. This work was supported by the University of Kurdistan.

Author Contribution Statement

GM conceived and designed research. GM and FG conducted data analysis and wrote the

manuscript.

Conflict of interest The authors declare that they have no competing interests.

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Tables and figure legends

Table 1 Information and physicochemical characteristics of PAO genes in bread wheat, T.

urartu and Ae. tauschii. Notes: AA, amino acid sequence length; MW, molecular weight; pI,

isoelectric point. ASN: alternative splice variants. “1” indicates only a single transcript. *:

wheat PAO genes that are confidently orthologous with the corresponding rice PAOs. ASN:

alternative splice variants from EnsemblPlants. D: gene direction, ‘+’: forward. ‘-’: reverse.

ASN**: alternative splice variants identified in ‘Manitou’ cultivar from experiment

SRP043554.

Species Name Transcript ID AA MW (kDa) pI ASN D ASN** TuPAO5 TRIUR3_11268-T1 520 57376.68 5.34 1 + - TuPAO9 TRIUR3_14057-T1 504 56367.99 6.45 1 + - T. urartu TuPAO6 TRIUR3_12020-T1 454 50979.07 7.12 1 + - TuPAO3 TRIUR3_18876-T1 484 53632.17 5.34 1 - - TuPAO4 TRIUR3_11269-T1 520 57376.68 5.34 1 + - TuPAO10 TRIUR3_14834-T1 490 55178.33 5.36 1 + - AetPAO4-2D AET2Gv21199400.1 490 53358.12 5.36 10 + - AetPAO3-2D AET2Gv21031900.5 513 57023.23 5.51 36 - - AetPAO5-2D AET2Gv21199100.12 492 54373.33 5.51 15 + - Ae. tauschii AetPAO9-4D AET4Gv20654900.7 526 59025.88 6.55 12 - - AetPAO6-7D AET7Gv21301800.1 498 55934.48 5.99 6 - - AetPAO11-7D AET7Gv20928100.8 503 56533.91 5.64 11 - - AetPAO1-3D AET3Gv20612000.2 517 55184.35 5.09 3 + - AetPAO10-4D AET4Gv20866800.1 245 26562.27 5.79 10 + - TaPAO8-1A TraesCS1A02G407600.1 585 61964.34 7.93 1 + 3 TaPAO8-5B TraesCS5B02G529400.1 585 62050.42 8.40 1 - 4 TaPAO8-5D TraesCS5D02G528500.1 582 61688.97 7.59 1 - 2 TaPAO10-4B TraesCS4B02G385300.1 481 53676.73 5.76 1 - 1 TaPAO10-5A TraesCS5A02G549600.1 495 55509.74 5.60 1 + 1 TaPAO11-7A TraesCS7A02G378800.1 457 51891.59 5.55 1 - 1 TaPAO11-7B TraesCS7B02G280700.1 477 54210.02 5.55 1 - 2 TaPAO11-7D TraesCS7D02G375700.1 503 56533.91 5.64 2 - 2 TaPAO2-2A TraesCS2A02G053400.1 340 37651.87 5.02 1 - 1 TaPAO3-2A* TraesCS2A02G467300.1 484 53646. 20 5.34 1 - 2 TaPAO3-2B* TraesCS2B02G490100.1 484 53604.16 5.34 1 - 3 TaPAO3-2D* TraesCS2D02G467300.1 484 53632.17 5.34 1 - 2 T. aestivum TaPAO4-2A* TraesCS2A02G548200.1 490 53266.07 5.37 1 + 2 TaPAO4-2B* TraesCS2B02G579100.1 490 53312.05 5.35 1 + 1 TaPAO4-2D* TraesCS2D02G549300.1 540 58748.37 5.64 1 + 2 TaPAO5-2A* TraesCS2A02G548100.1 487 53768.67 5.44 1 + 4 TaPAO5-2B* TraesCS2B02G579000.1 526 57604.88 5.45 1 + 2 TaPAO5-2D* TraesCS2D02G549200.1 492 54373.33 5.51 3 + 4 TaPAO6-7A* TraesCS7A02G539200.1 508 56928.63 6.58 2 - 2 TaPAO6-7B* TraesCS7B02G461800.1 495 55486.12 6.40 1 + 1 TaPAO6-7D* TraesCS7D02G524900.1 498 55946.45 5.99 1 + 1 TaPAO9-2A TraesCS2A02G159500.1 474 49845.89 6.27 1 + 1 TaPAO9-2B TraesCS2B02G185100.1 471 49635.65 6.11 1 + 1 TaPAO1-3A* TraesCS3A02G250700.1 510 54902.17 5.49 1 + 1 TaPAO1-3B* TraesCS3B02G280200.1 507 54518.62 5.22 1 + 1 TaPAO1-3D* TraesCS3D02G251100.1 491 52509.23 5.07 1 + 1 TaPAO7-4A TraesCS4A02G039600.1 468 52554.66 9.30 3 + 3 TaPAO7-4B TraesCS4B02G265900.1 493 55334.93 7.23 1 - 1 TaPAO7-4D TraesCS4D02G265800.1 493 55354.78 6.52 1 - 1 TaPAOUn TraesCSU02G062000.1 585 61995.31 7.58 1 + 1

Figure legends

Fig. 1 Polyamine biosynthesis in plants. ADC, arginine decarboxylase; AIH, agmatine

iminohydrolase; CPA, N-carbamoyl putrescine amidohydrolase; dcSAM: decarboxylated S-

adenosylmethionine; SAM: S-adenosylmethionine; SAMDC: S-adenosylmethionine

decarboxylase; SPDS: spermidine synthase; SPMS: spermine synthase; TSPMS:

thermospermine synthase; spermidine synthase: SPDS; spermine synthase: SPMS; PAO:

polyamine oxidase. The donor of the aminopropyl groups is dc-SAM, which is formed by

decarboxylation of SAM, through an enzymatic reaction catalyzed by SAMDC. The

aminopropyltransferases donating aminopropyl residue to Put or Spd for production of Spd or

Spm are SPDS and SPMS.

Fig. 2 Phylogenetic tree of PAO proteins from T. aestivum, T. urartu and Ae. tauschii, O. sativa

and A. thaliana.

Fig. 3 Chromosomal location of PAO genes on wheat chromosomes. Homoeologous genes

were mapped to 16 wheat chromosomes (composed of A, B, and D subgenomes) plus one

unassembled chromosome (Un) using shinyCircos. Homoeologs were connected using central

links. Chromosome were banded according to pTa535-1 (red bands) and (GAA)10 (blue bands)

FISH patterns. Chromosome number is indicated outside the outer circle.

Fig. 4 Gene structure, protein domain and motif analysis of TaPAOs. A) Exon–intron

structures of TaPAO genes. B) Distribution of conserved domains within TaPAO proteins. C)

Distribution of all motifs identified by MEME.

Fig. 5 Multiple sequence alignment of wheat and rice PAO protein sequences. The locations

and logos of the conserved domains of TaPAO genes identified by MEM are indicated. 29

Searching in Pfam identified domains 1, 2, 3 and 4 as Flavin containing amine oxidoreductase;

domain 6 as NAD_binding_8 and no result was found for domain 5.

Fig. 6 Log2 based expression levels for several TaPAO genes in different tissues during

developmental stages. TPM values belong to Ramírez-González, Borrill (42) and retrieved

from www.wheat-expression.com.

Fig. 7 Barplots of the transcript expression rates (mean ± sd) of TaPAO genes in common

wheat under different stress conditions including A) Leaf of ‘Manitou’ cultivar under normal

(control) and cold stress conditions. B) ‘Chinese Spring’ cultivar 4 days after mock inoculation

or inoculation with F. graminearum. C) Coleoptile-sheath-enclosed shoot tissue of common

wheat ‘Chara’, 3 days after mock inoculation or inoculation with F. graminearum. D, E and F)

seedlings of ‘TAM 107’ cultivar under a combined of heat and drought stress (40 °C and 20 %

PEG-6000) and normal (22 °C) conditions (D) heat (40 °C) and normal (22 °C) conditions (E)

and drought (20 % PEG-6000) and control (22 °C) conditions (F). G and H) Leaf tissue of

‘Ciza 168’ and ‘Gemmiza 10’ under control and PEG treatment conditions. I) Leaves of the

“Riband” cultivar after mock inoculation (control) or inoculation with Zymoseptoria tritici

isolate IPO323. J) Seedlings of the “Chinese Spring” cultivar 10 days after phosphorus

starvation and under control conditions. K) Seedlings of the “N9134” cultivar 7 days after

mock inoculation or after inoculation with powdery mildew. L) Seedlings of the “N9134”

cultivar seven days after mock inoculation or inoculation with stripe rust. In each experiment

‘*’, ‘**’ and ‘***’ indicate statistically significant differences from control at 0.05, 0.01 and

0.005 significant levels, based on DESeq2 adjusted p-values. M, N and O) Ternary plot

showing relative expression abundance of TaPAO genes under different stress conditions. In

each ternary plot, a circle or a triangles reflects the relative contribution of homoeologs of a

gene under the normal or stress condition respectively, and their sizes indicate the total 30

expression in TPM. The data code for each study and the evaluated wheat cultivar are also

indicated at the top (in barplots) or bottom (in ternary plots) of the subfigures.

Fig. 8 Expression levels in FPKM and the structure of distinct isoforms of the three TaPAO5-

2D genes under normal (23°C) and stress (4°C) conditions from the SRP043554 experiment

(A). Expression levels of isoforms are shown by barplots ± standard deviations (B) and in

varying shades of yellow (C). Boxes represent exons and horizontal lines connecting exons

represent introns.

O O O Glutamate Ornithine O O NH2 OH HN2 NH O NH 2 HO N NH2 Arginine Agmatine H ADC NH2 NH

AIH ODC Exapmle of TC pathway: O Methionine ZmPAO1, HvPAO1, HvPAO2, OsPAO2, NH2 CPA OsPAO6, OsPAO7 HO NH O2 +H 2 O 2 2 3 γ-aminobutyric acid N N (GABA) N Putrescine DAO N OH SAM O Δ-Pyrroline H C OH 3 HO2 2 SAMDC Example of BC pathway: OsPAO1, OsPAO3, 4‐aminobutanal O OsPAO4 and OsPAO5,

SPDS PAO O AtPAO (1-5) dc-SAM NH2 Spermidine PAO N N N O +H O N OH 2 2 HO2 2 HO2 2 1,3‐diaminopropane (DAP) O N‐(3‐aminopropyl)‐4‐aminobutanal

H3C + OH TSPMS/ACL5 SPMS S PAO 4‐aminobutanal H O O2 +H 2 O HO2 2 HN2 PAO 2 2 T-Spermine Spermine PAO cytoplasm enzymes chloroplast enzymes mitochondrial enzymes apoplast, cytoplasm, peroxisome or vacuole enzymes

Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.189209; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

III

AetPAO1−3D TaPAO1 −

3B 3A II AetPAO4−2D OsPAO1

AtPAO5 5B

TaPAO4− 3D

TaPAO1− TaPAO4− TaPAO1−

TaPAO2−2A

2D TaPAO8− 95 TaPAO8−5D TuPAO4 2B TaPAO4−2A 100 TaPAOUn 80 2B TaPAO8−1A IV 0 43 100 2A OsPAO4 100 33 TaPAO9− 3 100 TaPAO9− TuPAO5 99 100 100 7 100 TaPAO11−7A I TaPAO5−2D 100 39 TaPAO11−7B TaPAO5−2B 39 100 TaPAO11−7D 50 TaPAO5− 8 99 100 2A 89 AetPAO11−7D 100 3 AetPAO5−2D 100 76 57 100

OsPAO5 100 clade

TaPAO6−7B AtPAO4 100 100 100 100 TaPAO6− AtPAO2 7D 95 AetPAO6−7D AtPAO3 100 25 48 63 100 94 TaPAO6− 7A 61 100 TuPAO6 AetPAO3−2D 73 TaPAO7−4A TuPAO3 70 TuPAO7

100 TaPAO7− TaPAO3−2D 72 100 TaPAO7−4D V 100 TaPAO3−2B AetPAO7−4D TaPAO10−4B 4B AetPAO10−4D

TaPAO10−5A TuPAO10 species TaPAO3−2A OsPAO3 Transcript AtPAO1 variants Ae. tauschii OsPAO6 1 OsPAO7

A. thaliana OsPAO2 10 O. sativa VII 20 T. aestivum VI 36 T. urartu

Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.189209; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Chr 1A Chr Un 400MB

0MB 0MB Chr 1B 400MB 400MB

Chr 7D 0MB

T TaPAO6−7D aPAO1 TaPA 400MB 0MB Chr 1D

OUn O8−1A 0MB 1−7D A 400MB TaPA Chr 7B T TaP aP O6−7B A 400MB O11−7B

0MB 0MB

Chr 2A TaP A O2−2A 400MB TaP O6−7A A O9−2A AO1 aP A 400MB Chr 7A T 1−7A TaP

0MB

0MB TaPAO3−2AO5−2A 400MB TaPA O4−2A TaPA Chr 2B AO9−2B TaP 400MB Chr 6D

0MB

TaPAO3−2B TaPAO5−2B 800MB O4−2B 0MB (GAA)10 TaPA 400MB pTa535-1

Chr 6B A subgenome B subgenome 400MB TaPA Chr 2D 0MB D subgenome O3−2D TaPAO5−2D TaPAO4−2D

Chr 6A 400MB 0MB

TaP AO1−3A 0MB 400MB AO8−5D TaP Chr 3A

400MB Chr 5D 0MB TaP AO1−3B AO8−5B

0MB TaP 400MB

TaP Chr 3B 400MB Chr 5B A O1−3D 800MB TaPA 0MB TaPAO10−5A 0MB O7−4A

400MB

O7−4D A

400MB O7−4B aP 0MB Chr 5A O10−4B Chr 3D T

0MB aPA 400MB TaPA

0MB

400MB Chr 4D 0MB 400MB Chr 4A Chr 4B

Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.189209; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A B C TaPAO2-2A TaPAO1-3D TaPAO1-3A TaPAO1-3B TaPAO3-2A TaPAO3-2B TaPAO3-2D TaPAO5-2B TaPAO5-2A TaPAO5-2D TaPAO4-2B TaPAO4-2D TaPAO4-2A TaPAO11-7A TaPAO11-7B TaPAO11-7D TaPAO6-7B TaPAO6-7D TaPAO6-7A TaPAO10-5A TaPAO10-4B TaPAO7-4B TaPAO7-4D TaPAO7-4A TaPAO9-2B TaPAO9-2A TaPAO8-5D TaPAO8-5B TaPAO8-1A TaPAO-Un 3' 5' 0kb 1kb 2kb 3kb 4kb 5kb 6kb 7kb 8kb 9kb 10kb 0 100 200 300 400 500 600 0 100 200 300 400 500 600 Legend: Amino_oxidase NAD_binding_8 DAO Motif 6 Motif 3 Motif 5 CDS Intron ﬁve_prime_utr start_codon stop_codon three_prime_utr NAD_binding_9 FAD_binding_2 FAD_binding_3 Motif 2 Motif 4 Motif 1

Figure 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.189209; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Motif 6 Motif 3

TaPAO6-7B/1-495 1 ------MKPCTAIALLLVVAAQCAALATAAG--PRVI IVGAGMSGISAGKRLSEAG--ITDLVILEATDRIGGRIHKTKFAGVNVEMGANWVEGVNGDEMNPIWTMANGTGGLNLR 106 TaPAO6-7D/1-498 1 ------MLEMKPCRAIALALVVAAQCASLATAAG--PRVI IVGAGMSGISAGKRLSEAG--ITDLVILEATDRIGGRIHKTKFAGVNVEMGANWVEGVNGDEMNPIWTMANGTGGLNLR 109 TaPAO6-7A/1-508 1 ------MLEMKPCRAIALVLVVAAQCAALATAAG--PRVI IVGAGMSGISAGKRLSEAG--ITDLLILEATDRIGGRIHKTKFAGVNVEMGANWVEGVNGDEMNPIWTMANGTGGLNLR 109 TaPAO11-7B/1-477 1 ------MYLRREE------MGAGTAGISAGKRLWDAG- -VRDLL ILEATERVGGRMHKHNFGGLNVEIGA NWVEGLNGDKVNP IWPMVNAS- - LKLR 81 TaPAO11-7D/1-503 1 ------MKSWFVTAIAALVLIAAQHASLVAAGRG--PRVI IVGAGMSGISAGKRLWDAG- -VRDLL ILEATGRVGGRMHKHNFGGLNVE IGANWV EGLNGDKVNP IWPMVNAS- - LKLR 107 TaPAO11-7A/1-457 ------MSGISAGKRLWDAG- - LRDLL ILEATERVGGRMHKHNFGGLNVEIGANWVEGLNGDKVNP IWPMVNAS- - LQLR 70 TaPAO10-4B/1-481 1 ------MTPTGATAALVLAVLTLAHHGSLAAAAGGGPRVI IVGAGMSGISAGKRLSEAG- - ITDLV ILEATDHVGGRMHKRDLSGIKVEMG ------VNGGKMNP IWP IVNST- - LKLT 103 TaPAO10-5A/1-495 1 ------MTPTGATAALVLAVLTLAHHASLAAAGG--PRVI IVGAGMSGISAGKRLSEAG- - ITDLV ILEATDHVGGRMHKRDFGGISVEMGA NWVEGVNGGKMNP IWP IVNST- - LKLT 107 TaPAO7-4B/1-493 1 ------MTPT-IATALVLA-LTLAHHASLAAAAG--PRVI IVGAGMSGISAGKRLSEAG- - ITDLV ILEATDHVGGRMRKHNFGGVSVEI GANWVEGVNGGKMNP IWPLVNST- - LKLR 105 TaPAO7-4D/1-493 1 ------MTPT-ATTALVLA-LTLAHHASLAAAAG--PRVI IVGAGMSGISAGKRLSEAG- - ITDLV ILEATDHLGGRMRKHNFGGVSVEI GANWVEGVNGGKMNP IWPLVNST- - LKLR 105 TaPAO7-4A/1-468 1 ------MTPT-IATALVLA-LTLAHHASLASAAG--HRVI IVGAGMSGISAGKRLSEAG- - ITDLL ILEATDHVGGRMRKRNFGGVNVEMGA NWVEGVNGGKMNP IWPLVNST- - LKLR 105 OsPAO6/1-496 1 ------MTKPTTMAIFLVLALSIAQLLPSLVAGTGR-PRVI IVGAGISGISAGKR IWEAG- - IADVL ILEATDR IGGRMHKQSFAGVNV EIGANWVEGVNGEKKNP IWP IVNST- - LKLR 109 OsPAO7/1-474 1 ------MTKPTTMAIFLSIVLLSMAQLPSLVAGTGR-PRVI I IGAGISGISAGKRLSEAG- - ITDIL ILEATDHIGGRMHKQRFAGVNVEIGANWVEGVNGEKMNP IWP IVNST- - LKLR 109 OsPAO2/1-202 ------KLR 3 TaPAO3-2B/1-484 1 ------MANNNSSYGENVSRKSHTPSAI VIGGGFAGIAAANALRNAS---FEVVLLESRDRIGGRVHTDYSFGFPVDLGASWLHGVCEENPLAPIIGRLGLPLYRTS 98 TaPAO3-2D/1-484 1 ------MANNNSSYGENVSRKSHTPSAI VIGGGFAGIAAANALRNAS---FEVVLLESRDRIGGRVHTDYSFGFPVDLGASWLHGVCEENPLAPIIGRLGLPLYRTS 98 TaPAO3-2A/1-484 1 ------MANNNSTYGENVSRKSHTPSAI VIGGGFAGIAAANALRNAS---FEVVLLESRDRIGGRVHTDYSFGFPVDLGASWLHGVCEENPLAPIIGRLGLPLYRTS 98 OsPAO3/1-484 1 ------MANN-SSYGENVRRKSHTPSAI VIGSGFAGIAAANALRNAS---FEVVLLESRDRIGGRIHTDYSFGFPVDLGASWLHGVCEENPLAPIIGRLGLPLYRTS 97 TaPAO4-2B/1-490 1 ------MD--PNSFKTGGLLLPTIERRCASPPSVI VIGGGISGIAAARVLSNSS---FEVTVLESRDRIGGRVHTDYSFGCPIDMGASWLHGVSNENSLAPLIGHLGLRLYHTS 103 TaPAO4-2D/1-540 1 MSCLTFRRSAADASRLQDGLLVFMFFISIPIRSLNTSSSSSRRATVAPAAMD--PNSFKTGGLLLPTIERRCASPPSVI VIGGGISGIAAARVLSNSS---FEVTVLESRDRIGGRVHTDYSFGCPIDMGASWLHGVSNENSLAPLISHLGLRLYHTS 153 TaPAO4-2A/1-490 1 ------MD--PNSFKTGGLLLPTIERRCASPPSVI VIGGGISGIAAARVLSNSS---FEVTVLESRDRIGGRVHTDYSFGCPIDMGASWLHGVSNENSLAPLIGHLGLRLYHTS 103 OsPAO4/1-487 1 ------MD--PNSLKTGGLLLPTIERQCASPPSVI VIGGGISGVAAARALSNAS---FEVTVLESRDRVGGRVHTDYSFGCPIDMGASWLHGVCNENSLAPLIGYLGLKLYRTS 103 TaPAO5-2B/1-526 1 ------MRGDLREAPSGTTAAAAAAAVALQLGSSDRAPPRMDQPPNGFAAGGLFVQHIDGQNASPPSVI VIGGGISGIASARALSNAS---FKVTLLESRERLGGRVHTDYSFGCPIDMGASWLHGVCNENSLAPLIRLLGLRLYRTS 139 TaPAO5-2D/1-492 1 ------MDQPPNGFAAGGLFVQHIDGQKASPPSVI VIGGGISGIASARALSNAS---FKVTLLESRERLGGRVHTDYSFGCPIDMGASWLHGVCNENSLAPLIRLLGLRLYRTS 105 TaPAO5-2A/1-487 1 ------MASPPE-----GLFVQHIDGQNASPPSVI VIGGGISGIASARALSNAS---FKVTLLESRERLGGRVHTDYSFGCPIDMGASWLHGVCNENSLAPLIRLLGLRLYRTS 100 OsPAO5/1-492 1 ------MDQPSNGFAAGGLFLRHIDGQNASPPSVI VIGGGISGIAAARALSNAS---FKVTLLESRDRLGGRVHTDYSFGCPIDMGASWLHGVCNENSLAPLIRLLGLRLYRTS 105 TaPAO1-3A/1-510 1 ------MRARLARMIEKKPRIA IVGAGIAGLSAAQQLCGAGRDKFEVVVVEAGCRTGGRVFTSEFAGHRLEMGATWVQGIVGSPVYALAREAGALR-QEAA 94 TaPAO1-3B/1-507 1 ------MVEKKPRVV IVGAGIAGLSAAQQLCGAGRDKLEVVVVEAGCRAGGRVYTSEFAGHRLEMGATWVQGIVGSPVYALAREAGALR-QEAA 87 TaPAO1-3D/1-491 1 ------MAEKKPRIV IVGAGIAGLSAAQQLCGAGRDKFEVVVVEAGCRAGGRVYTSEFAGHRLEMGATWVQGIVGSPVYAMAREAGALR-EEAA 87 OsPAO1/1-512 1 ------MVAKKPRVV VVGAGISGLAAAHRLCGAGGDRFEVAVVEAGDRVGGRILTSEFAGHRVEMGATWVQGVVGSPVYALARDAGALGEEEGR 88 TaPAO8-1A/1-585 1 ------MASSALASPAPRPAPRGNRHPRACPAAAAAASPVPRRLGAAARCRAVAAPAGPSPPGVAERPEADVV VIGSGLGGLCCAGLLARYG---QDVVVLESHDRPGGAAHSFDVKGFHFDSGPSLFSGFQSRGPQANPLAQVLDALGESV 143 TaPAOUn/1-585 1 ------MASSALASPAPRPAPRGNRHPRACPAAAAAASPVPRRLGAAARCRAVAAPAGPSPPGVAERPEADVV VIGSGLGGLCCAGLLARYG---QDVVVLESHDRPGGAAHSFDVKGFHFDSGPSLFSGFQSRGPQANPLAQVLDALGESV 143 TaPAO8-5D/1-582 1 ------MASSALASPAPRPPPPGNRQPRARP---AAAAPVPRRVGAAARCRAVAAPAGPSPPGVAERPEADVV VIGSGLGGLCCAGLLARYG---QDVVVLESHDRPGGAAHSFDVKGFHFDSGPSLFSGFQSRGPQANPLAQVLDALGESV 140 TaPAO8-5B/1-585 1 ------MASSALASPSPRPPPRGNRQPRARPAAAAAAAPVPRRVGAAARCRAVAAPAGPSSPGVAGRPEADVV LIGSGLGGLCCAGLLARYG---QDVVVLESHDRPGGAAHSFDVKGFHFDSGPSLFSGFQSRGPQANPLAQVLDALRESV 143 TaPAO9-2A/1-474 1 ------MRVHAAHLLVPAP------PTPRRHATLR--FAVSAAATSVNVNQADAAGKQAV IVGGGLAGLAAATHLASLS---VPFTLLEASDRVGGRVATDEVDGYLLDRGFQIFL------TAYPECQRLLDFQALR 115 TaPAO9-2B/1-471 1 ------MRVHAAHLLVPAP------PTPRRHATLR--FAVSAAATSVNVNQAD---KQAV IVGGGLAGLAAATHLASLS---VPFTLLEASDRVGGRVATDEVDGYLLDRGFQIFL------TAYPECQRLLDFQALR 112 TaPAO2-2A/1-340 1 ------MRVA VVGAGVSGLAAAQELAASG--GARVTVYEKEDWLGGCARR----TVAVEDGAGLVHLDLCPMVFKQATCPNMMQLLEFL 77 Motif 5

TaPAO6-7B/1-495 107 T---FRSDFDHLASNTYKQDGGLYDEKVVEKIIE------RMDEVEESGSKLS GTLHHSGQQDMSVMAMQRLNDHMPSGP------ARPVDMVIDYYQHDFEFAEPPRVTSLQNTQPLPTFSNFGDDVYF 221 TaPAO6-7D/1-498 110 T---FRSDFDHLASNTYKQDGGLYDEKVVENIIE------RMDEVEESGSKLS GTLHHSGQQDMSVMAMQRLNDHMPSGP------ARPVDMVVDYYQHDFEFAEPPRVTSLQNTQPLPTFSNFGDDVYF 224 TaPAO6-7A/1-508 110 T---FRSDFDHLASNTYKQDGGLYDEKVVENIIE------RMDEVEESGSKLS GTLHHSGQQDMSVMAMQRLNDHMPSGP------ARPVDMVIDYYQHDFEFAEPPRVTSLQNTQPLPTFSNFGDDVYF 224 TaPAO11-7B/1-477 82 N---FYSDFDGVVGNVYKESGGLYDEEFVQKRMD------RGDEVEELGGKLA AKMDPSGRDDMSILAMQRLFNHQPNGP------ATPVDMVLDYFRYDYEFAEPPRVTSLQGTEPTATFADFGDDAHF 196 TaPAO11-7D/1-503 108 N---FYSDFDGVVGNVYKESGGLYDEEFVQKRMD------RGDEVEELGGKLA AEMDPSGRDDISILAMQRLFNHQPNGP------ATPVDMVLDYFRYDYEFAEPPRVTSLQGTEPTATFADFGDDANF 222 TaPAO11-7A/1-457 71 N---FYSDFDGVVGNVYKESGGLYDEEFVQKRMD------RGDEVEELGGKLA AKMDPSGRDDMSILAMQRLFNHQPNGP------ATPVDMVLDYFRYDYEFAEPPRVTSLQGTEPTATFADFGDDAHF 185 TaPAO10-4B/1-481 104 N---FRSDFDDLASNVYKEKGGVYKKAYVQKRID------RSDEVEEDGDKLS AKLHPSGQDDMSILAMQRLNDHLPNGP------SSPVDMILDYFKYDYEFAEPPRVTSLQNTVPLATFSDFGDDVYF 218 TaPAO10-5A/1-495 108 N---FRSDFDDLASNVYREKGGVYKKAYVQKRID------LSDKVQEGGEEHS AKLHLSGQDDMSILAMQRLNDHLPNGP------SAAVDMILDYFKYDYEFAEPPRVTSLQNTIPLATFNDFGDDVYF 222 TaPAO7-4B/1-493 106 N---FRSDFDGLAENVYKEKGGIYGRAYVQKRLD------RLDEVEEGGAKLP AKLRPSGQDDMSILAMQRLNNHMPNGP------ASPVDMILDYFKHDYEFAEPPRVTSLQNVVPLATFRDFGDDVYF 220 TaPAO7-4D/1-493 106 N---FRSDFDGLAENVYKETGGVYDRAYVQKRLD------RANEVEEGGAKLS AKLRPSGQDDMSILAMQRLNNHLPNGP------ASPVDMILDYFKHDYEFAEPPRVTSLQNVVPLATFRDFGDDVYF 220 TaPAO7-4A/1-468 106 N---FRSDFDGLAENVYKERGGVYDRAYVQKRLH------RSNEVEEGGAKLS AKLRPSGQDDMSILTMQRLNNHLPNGP------ASPVDMILDYFKHDYEFAEPPRVTSLQNVVPLATFRDY---VYF 217 OsPAO6/1-496 110 S---FRSDFDSLAQNVYK-DGGLCDEAYVQKRMD------RADEVDKSGENLS ATLHPSGRDDMSILSMQRLNDHLPNGP------SSPVDMAVDYFTYDYEFAEPPRVTSLQNTVPLPTFTDFGDDTYF 223 OsPAO7/1-474 110 N---FLSDFDSLAQNVYK-DGGLCDAAYVQKRID------LADEADKSGENLS ATLHPSGRDDMSILSMQRLNNHLPNGP------SSPVDMVVDYFTYDYEFAEPPRVTSLRNTVPLPTFTDFGDDNYF 223 OsPAO2/1-202 4 N---FRSDYDYLSQNVYKEDGGVYDEAQAQKIID------RAHEVNENGEKLS ATLPPSGRDDMSILTMQRLNDHLPNGP------SSPVDMVVDYYLYDYEYAEPPRVTSLQNAVPQRTFSDFGDDVYF 118 TaPAO3-2B/1-484 99 GDDSVLFDHDLESYALYDTNGSQVPQEFVEKIGK------VFEAILEETGKLR EEMK---EDISIAKAIAIVLEKNPHLR------QEGIAHDVLQWYLCR-MEGWFATDADAISLQCWDQEVLLPGGHGL 213 TaPAO3-2D/1-484 99 GDDSVLFDHDLESYALYDTNGSQVPQEFVEKIGK------VFEAILEETGKLR EEMK---EDISIAKAIAIVLERNPHLR------QEGIAHDVLQWYLCR-MEGWFATDADAISLQCWDQEVLLPGGHGL 213 TaPAO3-2A/1-484 99 GDDSVLFDHDLESYALYDTNGSQVPQEFVEKIGK------VFEAILEETGKLR EEMK---EDISIAKAIAIVLERNPHLR------QEGIAHDVLQWYLCR-MEGWFATDADAISLQCWDQEVLLPGGHGL 213 OsPAO3/1-484 98 GDDSVLFDHDLESYALYDTKGHQVPQELVEKIGK------VFETILEETGKLR EETK---EDISIAKAIAIVMERNPHLR------QEGIAHDVLQWYLCR-MEGWFATDADAISLQGWDQEVLLPGGHGL 212 TaPAO4-2B/1-490 104 GDNSVLYDHDLESCSLFDKNGVAVPMETAAKVGV------IFEKILKETVKLR DEQE---HDMPLEQAISMVLERHPDLK------LEGLDDRILQWCVCR-LEAWFAADADEISLKNWDQEHVLTGGHGL 218 TaPAO4-2D/1-540 154 GDNSVLYDHDLESCSLFDKNGLAVPMETAAKVGV------IFEKILKETVKLR DEQE---HDMPLEQAISMVLERHPDLK------LEGLDDRILQWCVCR-LEAWFAADADEISLKNWDQEHVLTGGHGL 268 TaPAO4-2A/1-490 104 GDNSVLYDHDLESCSLFDKNGLAVPMETAAKVGV------IFEKILKETVKLR DEQE---HDMPLEQAISMVLERHPDLK------LEGLDDRILQWCVCR-LEAWFAADADEISLKNWDQEHVLTGGHGL 218 OsPAO4/1-487 104 GDNSVLYDHDLESYALFDKAGHQVSKETVAKVEE------TFERILDETVKVR DEQE---HDMPLLQAISLVLERHPHLK------LQGIDDQVLQWCVCR-LEAWFAADADEISLKNWDQEHVLTGGHGL 218 TaPAO5-2B/1-526 140 GDNSVLYDHDLESYTLFDKDGRQIPQEIVTKVGE------IFEQILKETVKVR DEYA- - -NDMPLVQA ISMVLDRNPHLK------LEGLQYEVLQWC ICR- LEAWFATDVDNISLKNWDQEHVLTGGHGL 254 TaPAO5-2D/1-492 106 GDNSVLYDHDLESYALFDKDGRQIPQEIVTKVGE------IFEQILKETVKVR DEYA- - -NDMPLVQA ISMVLDRNPHLK------LEGLQYEVLQWC ICR- LEAWFATDVDNISLKNWDQEHVLTGGHGL 220 TaPAO5-2A/1-487 101 GDNSVLYDHDLESYALFDKDGRQIPQEIVTKVGE------IFEQILKETVKVR DEYA- - -NDMPLVQA ISMVLDRNPHLK------LEGLQYEVLQWC ICR- LEAWFATDVDNISLKNWDQEHVLTGGHGL 215 OsPAO5/1-492 106 GDNSVLYDHDLESYALFDKDGRQVPQEIVTKVGE------TFEKILKETVKVR AEHE---DDMPLIQAISIVLDRNPHLK------LDGLQYEVLQWCICR-LEAWFATDVDNISLKNWDQEHVLTGGHGL 220 TaPAO1-3A/1-510 95 DLPYERMDGFPDSVLTVAEGGDLVDADTVAKPIE------ELYRGMMEAARAG EAVGGGGVEEYLRRGLRAYQAARPGGS-----KEQEEIEEALIGMHINRERTDTSANDLGDLDLVAEGEYSDFPGDHVT 215 TaPAO1-3B/1-507 88 DLPYERMDGFPDSVLTVAEGGDVVDAVTVAKPIE------ELYRDMMEAARAG EAVGGGGVEDYLRRGLRAYQAARPGGS-----KELEEVEQALLVMHINRERTDTSADDLGDLDLAAEGEYRDFPGDHVT 208 TaPAO1-3D/1-491 88 DLPYERMDGFPDSVLTVAEGGDVVDADTVAKPIE------DLYRGMMKAARAG EAVGGGGVEEYLRRGLRAYQAARPVGS-----KELEEIEEALLSMHINRERTDTSADDLGDLDLAAEGEYRDFPGDHVT 208 OsPAO1/1-512 89 GLPYERMDGFPDRVLTVAEGGEVVDADTVAGPIE------ELYRGMMEAARAG EAGGGGGVEEYLRRGLRAYQAARSAGGGGGGGKELEEVDEALLAMHINRERTDTSADDLGDLDLTAEGEYRDFPGEHVT 214 TaPAO8-1A/1-585 144 PCASYDSWMVHVPEGQFESRIGPTDFLKDLETYVGLDATREWQKLLDAVIPISAAAMALPPLSIRGDLGILSTAAGRYA PSLLQSLIKMGPRGALGATKLLRPFSEIVDSLELKNPFVRNWIDLLCFLLAGVKSDSALSAEMVYMFAEWYKPGC-KLE 300 TaPAOUn/1-585 144 PCASYDSWMVHVPEGQFESRIGPTDFLKDLETYVGLDATREWQKLLDAVIPISAAAMALPPLSIRGDLGILSTAAGRYA PSLLQSLIKMGPRGSLGATKLLRPFSEIVDSLELKNPFVRNWIDLLCFLLAGVKSDSALSAEMVYMFAEWYKPGC-KLE 300 TaPAO8-5D/1-582 141 PCASYDSWMVHVPEGQFESRIGPTDFLKDLETYVGLDATREWQKLLDAVIPISAAAMALPPLSIRGDLGILSTAAGRYA PSLLQSLIKMGPRGALGATKLLRPFSEIVDSLELKNPFVRNWIDLLCFLLAGVKSDSALSAEMVYMFAEWYKPGC-KLE 297 TaPAO8-5B/1-585 144 PCASYDSWMVHVPEGQFESRIGPTDFLKDLETYVGLDATREWQKLLDAVIPISAAAMALPPLSIRGDLGILSTAAGRYA PSLLQSLIKMGPRGALGATKLLRPFSEIVDSLELKNPFVRNWIDLLCFLLAGVKSDSALSAEMVYMFAEWYKPGC-KLE 300 TaPAO9-2A/1-474 116 LQPFFPGALVYIGAGEGGS---PFHMLSDPFRFP------IR-SLSSVFSPVGTLPDKLLVGITRLRAAATPDDVILSSPETTTARHLEKLGFSPSIVERFLRPFLAGIFFDPALDTSSRLFELVFKRLALGDNA 240 TaPAO9-2B/1-471 113 LQPFFPGALVYIGAGEGGS---PFHMLSDPFRFP------IR-SLSSVFSPVGTLPDKLLVGITRLRAAATPDDVIVSSPETTTATHLEKLGFSPSIVERFLRPFLAGIFFDPALDTSSRLFELVFKRLALGDNA 237 TaPAO2-2A/1-340 78 GLEIERTEIPFSVSTKLDNGSQCYWSTSNG------ISSLLAQKSNALQ PSFWCMIREILK------FKTDVLGYLDYYESNPDLDQNETLGRFVQSHEYSHLFQEAYL 174

Motif 1 Motif 5

TaPAO6-7B/1-495 222 VADQRGYESVVYHVAGQYLKTDRKSGAIVDPRLKLNTVVREISYFPSGVTVKTEDNKVYRADYVVVSASLGVLQTDL------IRFKPQLPSWKIVS IYQFDMAVYTKIF LKFPKRFWPEGTGKEFFLYASGRRGYFPVWEQFE------359 TaPAO6-7D/1-498 225 VADQRGYESVVYHVAGQYLRTDRKSGAVTDPRLKPNTVVREISYFPSGVTVKTEDDKVYRADYVVVSASLGVLQTDL------IRFKPQLPSWK IVS IYQFDMAVYTKVFLKFPKRFWPEGEGKEFFLYASGRRGYFPVWQQFE------362 TaPAO6-7A/1-508 225 VADQRGYESVVYHVAGQYLKTDRKSGAIVDPRLKLNTVVREISYFPSGVMVRTEDNKVYRADYVVVSASLGVLQTDL------IRFKPQLPSWK IVS IYQFDMAVYTK IF LRFPKRFWPEGSGKEFFLYASGRRGYFPVWQQFE------362 TaPAO11-7B/1-477 197 VADQRGFETLIYHIAGQYLRSD-KSGNIVDPRVKLNKVVREISYNQRGVVVTTEDNSAYSADYVIVSASIGVLQRDL------IQFKPQLPAWK IFA IYQFDMGVYTK IF LKFPRKFWPTGPGKQFFVYASSRRGYYGMWQSFE------333 TaPAO11-7D/1-503 223 VADQRGFETLIYHIAGQYLRSD-KSGNIVDPRVKLNKVVREISYDQRGVVVTTEDNSAYSADYVIVSASIGVLQSDL------IQFKPQLPAWK ILA IYRFDMGVYTK I FLKFPRKFWPTGPGKQFFVYASSRRGYYGMWQSFE------359 TaPAO11-7A/1-457 186 VADQRGFETLIYHIAGQYLRSD-KSGNIIDPRVKLNKVVREISYKQRGVVVTTEDNSAYSADYVIVSASIGVLQSDL------IQFKPQLPAWK ILA IYRFDMGVYTK I FLEFPRKFWPTGPGKQFFVYASSRRGYYGMWQSFE------322 TaPAO10-4B/1-481 219 VADQRGYEAVVYYLAGQYLKAD-KAGNIVDPRLQLNKVVREISYSTRGVEVKTEDNKVYKADYVMVSTSLGVLQSDL------I Q F K P Q L P T WK V L S I Y Q F DMA V Y T K I L S S S P R - - - - - S S GR - - - - K V K S QWNC Y - V I Q E F E ------345 TaPAO10-5A/1-495 223 VADQRGYEAVVYHLAGQYLKAD-KSGGIIDPRLQLNKVVREISYSGSGVRVETEDNKVYKADYVMVSTSLGVLQSDL------IKFEPQLPTWKVLS IYQFDMAVYTK IFVKFPKKFWPEGKGREFFLYASSRRGYYGVWQEFE------359 TaPAO7-4B/1-493 221 VADQRGYEAVVHHLAGQYLKAD-KSGDIVDPRLQLNKAQKEISHSGSGVAVKTEEKKVYKADYVMVSTSVGVLQSDL------IQFKPRLPAWK ILS IYQFDMAVYTK I FVKFPRKFWPEGKGREFFLYASSRRGYYGVWQEFE------357 TaPAO7-4D/1-493 221 VADKRGYEAVVHHLAGQYLEAD-KARNIVDPRLQLNKVVREISYSGSGVTVRTEDDKVYKADYVMVSTSVGVLQSDL------IQFKPRLPAWKVLS IYQFDMAVYTK IFVKFPRKFWPEGKGREFFLYASSRRGYYGVWQELE------357 TaPAO7-4A/1-468 218 VADRRGYEAIVHHLAGQYLEAD-KSGNIV------VREISHSGSGVAVKTEDDKVYKADYVMVSTSVGVLQSDL------IQFKPRLPAWK ILS IYQFDMAVYTK I FVKFPRKFWPEGKGREFFLYASSRRGYYGVWRRQR------345 OsPAO6/1-496 224 VADQRGYESVVHHLAGQYLNAD-KSGNIADARLKLNKVVREISYSSTGVTVKTEDNSTYQADYVMVSASLGVLQSDL------IQFKPQLPSWK ILA IYQFDMAVYTK I FVKFPKKFWPEGAGREFFLYASTRRGYYGVWQEFE------360 OsPAO7/1-474 224 VADQRGYEAVVYYLAGQYLEAD-KSGNIVDARLQLNKVVREISYSSTGVTVKTEDNSTYQADYVMVSASLGVLQSDL------IQFKPQLPSWK ILA IYQFDMAVYTK I FVKFPKKFWPEGAGREFFLYASTRRGYYGVWQEFE------360 OsPAO2/1-202 119 VADKRGYESVVHYLAGQYLNTD-DSGNVADPRLQLNKVVREISYSSSGVTVKTEDGSVYQADYRHGLCQLG------SPAERSYTVQATAA------202 TaPAO3-2B/1-484 214 MVR--GYRPVINTLAKGLD------IRLGHRVVEIVRHWNRVEVTVSNGKTFVADAAVITVPLGVLKSNT------IKFEPRLPEWKEEAIRELSVGVENKIVLHFSEVFWPNVEFLGVVSSTTYGCSY-FLNLHKA------335 TaPAO3-2D/1-484 214 MVR--GYRPVINTLAKGLD------IRLGHRVVEIVRHWNRVEVTVSNGKTFVADAAVITVPLGVLKSNT------IKFEPRLPEWKEEAIRELSVGVENKIVLHFSEVFWPNVEFLGVVSSTTYGCSY-FLNLHKA------335 TaPAO3-2A/1-484 214 MVR--GYRPVINTLAKGLD------IRLGHRVVEIVRHWNRVEVTVSNGKTFVADAAVITVPLGVLKSNT------IKFEPRLPEWKEEAIRELSVGVENKIVLHFSEVFWPNVEFLGVVSSTTYGCSY-FLNLHKA------335 OsPAO3/1-484 213 MVR--GYRPVINTLAKGLD------IRLGHRVVEIVRHRNRVEVTVSSGKTFVADAAVIAVPLGVLKANT------IKFEPRLPEWKEEAIRELSVGVENKIILHFSEVFWPNVEFLGVVSSTTYGCSY-FLNLHKA------334 TaPAO4-2B/1-490 219 MVD--GYFPVIQALAQGLD------IRLNQRVKKITRQHKGVTVTIEDGTQYSADACIITVPLGVLKANI------IKFEPELPSWKSSAIADLGVGIENKVAMHFDKAFWPNVQVLGMVGPTPKTCGY-FLNLHKA------340 TaPAO4-2D/1-540 269 MVD--GYFPVIQALAQGLD------IRLNQRVKKIARQQKGVTVTIEDGTQYSADACIITVPLGVLKANI------IKFEPELPSWKSSAIADLGVGIENKVAMHFDKAFWPNVQVLGMVGPTPKTCGY-FLNLHKA------390 TaPAO4-2A/1-490 219 MVD--GYLPVIQALAQGLD------IRLNQRVKKIARQQKGVTVTIEDGTQYSADACIITVPLGVLKANI------IKFEPELPSWKSSAIADLGVGIENKVAMHFDKAFWPNVQVLGMVGPTPKTCGY-FLNLHKA------340 OsPAO4/1-487 219 MVN--GYYPIIQALAQGLD------IRLNQRVTKIARQFNGVTVTTEDGTSYSADACIITVPLGVLKANI------IKFEPELPSWKSSA IADLGVGIENK IAMHFDTVFWPNVEVLGMVGPTPKACGY-FLNLHKA------340 TaPAO5-2B/1-526 255 MVN--GYDPVIKALSRDLD------VHLNHRVTKIIQRYNKVIVCVEDGTSFVADAAIITVPLGVLKANI------IKFEPELPDWKLSAISDLGVGLENKIALRFNTVFWPNVEVLGRVAQTSNACGY-FLNLHKA------376 TaPAO5-2D/1-492 221 MVN--GYDPVIKALSRDLD------VHLNHRVTKIIQRYNKVIVCVEDGTSFVADAAIITVPLGVLKANI------IKFEPELPDWKLSAISDLGVGLENKIALRFNTIFWPNVEVLGRVAQTSNACGY-FLNLHKA------342 TaPAO5-2A/1-487 216 MVN--GYDPVIKALSRDLD------VHLNHRVTKIIQRYNKVIVCVEDGTSFVADAAIITVPLGVLKANI------IKFEPELPDWKLSAISDLGVGLENKIALRFNTIFWPNVEVLGRVAQTSNACGY-FLNLHKA------337 OsPAO5/1-492 221 MVH--GYDPVIKALAQDLD------IHLNHRVTKIIQRYNKTIVCVEDGTSFVADAAIITVPLGVLKANI------IKFEPELPDWKLSSISDLGIGIENKIALRFNSVFWPNVEVLGRVAPTSNACGY-FLNLHKA------342 TaPAO1-3A/1-510 216 IPG--GYTRVVEHLVAALPPGT------VRLGLRLRRLDWGETAVRLHFADEATTTLTADHVILTVSLGVLKASIGK DVSAT------ADPPLPQFKRDAVARLGFGIVDKLFIDLEAVETPEPDGGDEQLARAASPAFPFLHMAFVGDVAKIP W 356 TaPAO1-3B/1-507 209 IPG--GYTRVVEHLAAALPPGT------VRLGLRLRRLDWGETPVRLHFADEATTTLTADHVILTVSLGVLKASIGE DVSAAG---AIAFDPPLPQFKREAVARLGFGVVDKLFIELEDVEMPEPDGDDEQQARTAPPTFPFLHMAFVGDVAKIP W 353 TaPAO1-3D/1-491 209 IPS--GYTRVVEHLVAALP------ARYLRLHFGDEATTTLTADHVILTVSLGVLKASIDEDVSATG---AIAFDPPLPEFKREAVARLGFGVVDKLFVEVEAVETPELDGGDEQLASAAPPAFPFLHMAFLGDAAKIP W 337 OsPAO1/1-512 215 IPG--GYSRVVERLAAALPPGT------VRLGLRLRRLKWGGTPVRLHFADGAPP-LTADHVILTVSLGVLKASLGN KDTAGVGAAAIAFDPPLPPFKREAVARLGFGVVNKLFMEVEAVAPSEPE--DVAGVQPAAAGFPFLHMAFRGHVSKIP W 359 TaPAO8-1A/1-585 301 YPLG-GSGAIIDALVRGIKK------FGGRLALNSHVEKILIENGRAVGVKLRGGQIVRAKKAVVSNASMWDTLG------LLPPDAVPRSYQDQVKATPQCDSFMHLHLGFDTECVKEDLGIHHIVVNDWDKGVDGEQNVVLISVPSVLS 438 TaPAOUn/1-585 301 YPLG-GSGAIIDALVRGIEK------FGGRLALNSHVEKILIENGRAVGVKLRGGQIVRAKKAVVSNASMWDTLG------LLPPDAVPRSYQDQVKATPQCDSFMHLHLGFDTECVKEDLGIHHIVVNDWDKGVDGEQNVVLISVPSVLS 438 TaPAO8-5D/1-582 298 YPLG-GSGAIIDALVRGIEK------FGGRLALNSHVEKILIENGRAVGVKLRGGQIVRAKKAVVSNASMWDTLG------LVPPDAVPRSYQDQVKATPQCDSFMHLHLGFDAECVKEDLGIHHIVVNDWDKGVDGEQNVVLISVPSVLS 435 TaPAO8-5B/1-585 301 YPLG-GSGAIIDALVRGIEK------FGGRLALNSHVEKILIENGRAVGVKLRSGQIVRAKKAVVSNASMWDTLG------LLPPDAVPRSYQDQVKATPQCDSFMHLHLGFDAECVKEDLGIHHIVVNDWDKGVDGEQNVVLISVPSVLG 438 TaPAO9-2A/1-474 241 LPEA-GIGAIAAQLADRLP------AGSVRLNARVAAIDPSS----GVTLDTGETVSGKLGVIVAVEQPEAEK------LLPQLPARPKNKKAAERSTVC-----LYFSADRAAVQEPV----LLLNGSGKGVVNN-----MFFATNVA 358 TaPAO9-2B/1-471 238 LPEA-GIGAIAGQLADRLP------AGSVRLNARAAAVDPSS----GVTLDTGETVSGELGVIVAVEQPEAEK------LLPQLPTRPKNKKAAERSTVC-----LYFSADRAAVQEPV----LLLNGSGKGIVNN-----MFFATNVA 355 TaPAO2-2A/1-340 175 IPICTSVWCCPSQGVLGFS------AVSVLSFFRNHDLFQLFGHPESFIVRGCLQSFVDKARAELER------MACRIKTSCTVKSVSCDDGGGYRVQEED------263

Motif 2 Motif 4

TaPAO6-7B/1-495 360 ------QQYPGSNVLLVTVTDDES------RRIEQQSDNQTMAEAVAVLRKMFP------GK DVPDATEILVPRWWSNRFFKGSFSNWPIGVNRYEYDL IRAPVG------RVYFTGEHTSEKYNGYVHGAYL 468 TaPAO6-7D/1-498 363 ------QQYPGSNVLLVTVTDDES------RRIEQQSDNQTMAEAVAVLRKMFP------KEDVPDATEILVPRWWSNRFFKGSFSNWPIGVNRYEYDL IRAPVG------RVYFTGEHTSEKYNGYVHGAYL 471 TaPAO6-7A/1-508 363 ------QQYPGSNVLLVTVTDDES------RRIEQQSDNQTMAEAVAVLRKMFP------GEDVPDATEILVPRWWSNRFFKGSFSNWPIGVNRYEYDL IRAPVG------RVYFTGEHTSEKYNGYVHGAYL 471 TaPAO11-7B/1-477 334 ------QEYPGANVLMATVTDVES------RRIEQQPDNVTMAEAVGVLRNMFP------DR DVPDATDIYVPRWWSNRFFKGSYSNWPVGVNRYEYDQLRAPVGG------RVYFTGEHTSEHYNGYVHGAYL 443 TaPAO11-7D/1-503 360 ------QEYPGANVLMVTVTDVES------RRIEQQPDNVTMAEAVGVLRNMFP------DR DVPDATDIYVPRWWSNRFFKGSYSNWPVGVNRYEYDQLRAPVGG------RVYFTGEHTSEHYNGYVHGAYL 469 TaPAO11-7A/1-457 323 ------QEYPGANVLMVTVTDVES------RRIEQQPDNVTMAEAVGVLRNMFP------DR DVPDATDIYVPRWWSNRFFKGSYSNWPVGVNRYEYDQLRAPVGG------RVYFTGEHTSERYNGYVHGAYL 432 TaPAO10-4B/1-481 346 ------AQYPDANVLLVTVTDEES------RRIEQQSDNQTKVEIMEVLRSMFP------GEDVPDATDILVPRWWSNRFYKGTFSNWP IGVNRYEYDQLRAPVG------RVYFTGEHTSEHYNGYVHGAYL 454 TaPAO10-5A/1-495 360 ------AQYPDANVLLVTVTNEES------RRIEQQSDNQTKAEIMEVLRSMFP------DEDVPDATDILVPRWWSDRFYMGTFSNWP IGVNRYEYDQLRAPVG------RVYFTGEHTNEHYNGHVHGAYL 468 TaPAO7-4B/1-493 358 ------AQYPDANVLLVTVTDEES------RRIEQQSDNQTKAEIVEVLRSMFP------GEDVPDATDILVPRWWSNRFYRGTFSNWPIGVNRYEYDQLRSPVG------RVYFTGEHTSEHYNGYVHGAYL 466 TaPAO7-4D/1-493 358 ------APYPDANVLLVTVTDEES------RRIEQQSDNQTKAEIVEVLRSMFS------GEDVPDATDILVPRWWSDRFYRGTFSNWP IGVNRYEYDQLRAPVG------RVYFTGEHTSEHYNGYVHGAYL 466 TaPAO7-4A/1-468 346 ------SPG-DRHRRGVEADRAAA------RQPDQGGD------RGVL------GEDVPDATDILVPRWWSDRFYRGTFSNWP IGVNRYEYDQLRAPVG------RVYFSGEHTSEHYNGYVHGAYL 441 OsPAO6/1-496 361 ------KQYPDANVLLVTVTDEES------RRIEQQPDSQTKAEIMEVVRCMFP------DEDVPDATDILVPRWWSDRFFRGSFSNWPIGVSRYEYDQLRAPVG------RVYFTGEHTSERYNGYVHGAYL 469 OsPAO7/1-474 361 ------KQYPDANVLLVTVTDEES------RRIEQQPDSQTKAEIMEVVRSMFP------DEDVPDATDILVPRWWSDRFFQGSFSNWPIGVSRYEHDQLRAPVG------RVYFTGEHTSERYNGYVHGAYL 469 OsPAO2/1-202 ------TaPAO3-2B/1-484 336 ------TGHAVLVYMPAGRLA------CDIEKMSDEAAAQFAFSQLKKILP------NAAEPLNYLVSHWGSDENTLGSYTFDGVGKPRDLYEKLRIPVDN------LFFAGEATSVQYTGTVHGAFS 439 TaPAO3-2D/1-484 336 ------TGHAVLVYMPAGRLA------CDIEKMSDEAAAQFAFSQLKKILP------NAAEPLNYLVSHWGSDENTLGSYTFDGVGKPRDLYEKLRIPVDN------LFFAGEATSVQYTGTVHGAFS 439 TaPAO3-2A/1-484 336 ------TGHAVLVYMPAGRLA------CDIEKMSDEAAAQFAFSQLKKILP------NAAEPLNYLVSHWGSDENTLGSYTFDGVGKPRDLYEKLRIPVDN------LFFAGEATSVQYTGTVHGAFS 439 OsPAO3/1-484 335 ------TGHPVLVYMPAGRLA------CDIEKLSDEAAAQFAFSQLKKILP------NAAEPIHYLVSHWGSDENTLGSYTFDGVGKPRDLYEKLRIPVDN------LFFAGEATSVQYTGTVHGAFS 438 TaPAO4-2B/1-490 341 ------TGHPVLVFMAAGRFA------QDVEKLSDKEAVELVMCHLRKMIP------DATEPTQYLVSRWGSDPNSLGSYSCDLVGKPSDVCERFSAPVESV------LYFAGEAASAEHSGAVHGAYS 445 TaPAO4-2D/1-540 391 ------TGHPVLVFMAAGRFA------QDVEKLSDKEAVELVMCHLRKMIP------DATEPSQYLVSRWGSDPNSLGSYSCDLVGKPSDVCERFSAPVESV------LYFAGEAASAEHSGAVHGAYS 495 TaPAO4-2A/1-490 341 ------TGHPVLVFMAAGRFA------QDVEKLSDKEAVELVMCHLRKMIP------DATEPNKYLVSRWGSDPNSLGSYSCDLVGKPSDVCERFSAPVESV------LYFAGEAASAEHSGAVHGAYS 445 OsPAO4/1-487 341 ------TGNPVLVYMAAGRFA------QEVEKLSDKEAVDLVMSHLKKMLP------DATEPTKYLVSRWGSDPNSLGSYSCDLVGKPADVSARFAAPVEN------LYFAGEAASADHSGSVHGAYS 444 TaPAO5-2B/1-526 377 ------TGHPVLVCMVAGRFA------YEMEKLSDEESVNFVMSQLRRMLP------GATEPVQYLVSRWGTDPNSLGSYSCDLVGKPADLYERFCAPVGN------LFFAGEAACIDHSGSVHGAYS 480 TaPAO5-2D/1-492 343 ------TGHPVLVCMVAGRFA------YEMEKLSDEESVNFVMSQLRRMLP------GATEPVQYLVSRWGTDPNSLGSYSCDLVGKPADLYERFCARVGN------LFFAGEAACIDHSGSVHGAYS 446 TaPAO5-2A/1-487 338 ------TGHPVLVCMVAGRFA------YEMEKLSDEESVNFVMSQLRRMLP------GATEPVQYLVSRWGTDPNSLGSYSCDLVGKPADLYERFCAPVGN------LFFAGEAACIDHSGSVHGAYS 441 OsPAO5/1-492 343 ------TGHPVLVCMVAGRFA------YEFEKLSDEESVNFVMSQLKKMLP------GATEPVQYLVSRWGTDPNSLGSYSCDLVGKPADLYERFCAPVGN------LFFAGEAACIDHSGSVHGAYS 446 TaPAO1-3A/1-510 357 WMRGTESVCPVHAASTVALAWFAGREA------AHLESLPDDEVIRAVHSTLESFLPAPPRRCGGAGAGPT PRWRVKR IKRSGWATDPLFLGSYSYVAVGSSGEDLDWMAEPLPRGQEAGR- - - - TPLRVLFAGEATHRTHYSTTHAAYL 496 TaPAO1-3B/1-507 354 WMRGTESVCPVHAGSTVALAWFAGREA------AHLESLPDDEVIRAVHSTLESFLPGPPRRCSGAGAGAT PRRRVRRIKRSGWATDPLFLGSYSYVAVGSSGEDLDRMAEPLPRGPEAGR----TPLSVLFAGEATQRTHYSTTHAAYL 493 TaPAO1-3D/1-491 338 WMRGTESVRPVHAGSTVALAWFAGREA------THLESLPDDEVIHALHSTLESFLPAPPRRCSGAAAGAT PRWRVKRIKRSGWATDPLFLGSYSYVAVGSSGEDLDRMAEPLPSGPEAGG----TPLSVLFAGEATHRTHCSTTHAAYL 477 OsPAO1/1-512 360 WMRGTESICPVHAGSTVALAWFAGREA------AHLESLPDDDVIRGAHATLDSFLP------AA PRWRVRRIKRSGWATDPLFLGSYSYVAVGSSGDDLDRMAEPLPRGPDAAADERPPSPRLLFAGEATHRTHYSTTHAAYL 491 TaPAO8-1A/1-585 439 EG------LAPPGKHILHAYTPGTEPFGLWDGLDRKSAAYQSLKEERSEVMWKAVELALGPKFSR------E TaPAOUn/1-585 439 EG------LAPPGKHILHAYTPGTEPFGLWDGLDRKSAAYRSLKEERSEVMWKAVELALGPKFSR------E KCEVKLVGTPLTHKRFLRRNRGTYGPAIKAGEATFPGQATPVPQ------LFCCGDSTFPGIG--VPAVAA 561 TaPAO8-5D/1-582 436 EG------LAPPGKHILHAYTPGTEPFGLWDGLDRKSAAYRSLKEERSEVMWKAVELALGPKFSR------E KCDVKLVGTPLTHKRFLRRNRGTYGPAIKAGEATFPGQATPVPQ------LFCCGDSTFPGIG--VPAVAA 558 TaPAO8-5B/1-585 439 EG------LAPPGKHILHAYTPGTEPFGLWEGLDRKSAAYRSLKEERSEVMWKAVELALGPKFSR------E KCEVKLVGTPLTHKRFLRRNRGTYGPAIKAGEATFPGQATPVPQ------LFCCGDSTFPGIG--VPAVAA 561 TaPAO9-2A/1-474 359 PS------YAPAGKVLVSVSLVG------SFADREDAELAGEVVRELGGWFG------AGEVASWAHLRTYRIGFAQPDQTPPTEPAGRDPRAGDG------VYVCGDHWCSAT---FDGAMV 454 TaPAO9-2B/1-471 356 PS------YAPAGKVLVSVSLVG------SFAGREDAELAGEVVRELGGWFG------AGEVASWAHLRTYRIGFAQPNQTPPTEPTGRDPRIGDG------VYVCGDHWCSAT---FDGAMV 451 TaPAO2-2A/1-340 264 ------GS------EEIYDKVILGIHAPAALKVLG------AEATHEELRILGAFQYVCSDAYLHCDKSLMPQN------LSGWSAWNFLGETSRDMFV 338

TaPAO6-7B/1-495 469 AG----IDSADILINCAKKKMCK---YDVKGKHD------495

TaPAO6-7D/1-498 472 AG----IDSADILINCAKKKMCK---YDVKGKHD------498 4 TaPAO6-7A/1-508 472 AVSTLLTSSSTVPRRRCANTMSRGSMTNLKVNHGVTS------508 2 TaPAO11-7B/1-477 444 AG----IDSADILINKVLNNVEFKVRPKYDDEQKAEAK------477 Motif 1 bits I VSVS QSD K K Q S I L QF M YT F K RK V L V D G A Y P V R A S S L S TaPAO11-7D/1-503 470 AG----IDSADILMNKVLNNVEFKVRPKYDEEQKAEAK------503 V A LV E SV D LV VH KR A A I Q D L RE G A R NTA T E VMS I C R T E F R S ILSES ESSGR 0 M T I T R E S A A I V D 2 3 4 5 6 7 8 9 1 I T P K N E L V EN TaPAO11-7A/1-457 433 AG----LLSSSGPSREVVCDLRCMRFFLC------457 GVL10 11 12 13 14 I 15 16 F17 18 P19 20 L21 P22 23 W24 K25 26 27 28 I29 30 31 32 33 34 35 36 37 38 K39 40 41 42 43 F44 45 46 47 F48 W49 P50 TaPAO10-4B/1-481 455 SG----IDSADILINCAQKSMCKYHVQGKYD------481 TaPAO10-5A/1-495 469 SG----IDSADILINCAQKSMCKYHVQGKYD------495 4 TaPAO7-4B/1-493 467 SG----IDSADILIKCAQKRMCKYHIPGKFD------493 2 Motif 2 bits D S W RFF NWP VN YE RL TaPAO7-4D/1-493 467 SG----IDSADILIKCAHKRMCKYHIPGKFD------493 Q I SL D G D Y V RS RS P R C Q L L LF AI Y H S L N L R G PNYK S YDG KPGDVY MC E E R K E TaPAO7-4A/1-468 442 SG----IDSADILIKCAHKRMCKHHIPGKFE------468 K KR G AT ELTM T STFVA SSAEDC W I ASRLD 0 N F I 1 Y2 3 V4 P5 6 7 8 9 E V 10 11 12 13 14 G15 16 17 18 19 20 21 22 G23 24 25 26 27 28 29 30 31 32 33 34 P35 36 37 OsPAO6/1-496 470 AG----IDSAEILINCAQKKMCKYNVGGKHG------496 W OsPAO7/1-474 470 AG----IYA------474 4 OsPAO2/1-202 ------TaPAO3-2B/1-484 440 TG----EMAAEECRMRVLEKFRELDMLEMCHP-MAEQTATVSVPLLISRL 484 2 I Motif 3 bits AT V V TDYFF FP D S VE N VV N V G H G VN M VC V R I F CH I I H FQN TaPAO3-2D/1-484 440 TG----EMAAEECRMRVLEKFRELDMLEMCHP-MAEQTATVSVPLLISRL 484 P MR DS L L D T A L S S T H S S V E HY L P L H N Q I A S SE G L K E K YR L ATW C A A D L F I LTAYP LFLLV SGRHT A I FSRKLVS I KF R VQGFGKMNP I 0 S R E G TaPAO3-2A/1-484 440 TG----EMAAEECRMRVLEKFRELDMLEMCHP-MAEQTATVSVPLLISRL 484 1 2 3 L4 5 6 7 8 9 E 10 GG11 12 13 14 15 16 17 18 19 20 G21 22 23 24 25 26 G27 28 29 30 31 32 33 34 35 36 OsPAO3/1-484 439 TG----LMAAEECRMRVLERFRELDMLEMCHPAMGEQTATVSVPLLISRL 484 TaPAO4-2B/1-490 446 SG----MDAAEECRRRLLMSKGVPDLIQVGAA-ACEEMADVVAPLQICRT 490 4 TaPAO4-2D/1-540 496 SG----MDAAEECRRRLLMRKGVPDLVQVGAA-ACEEMADVVAPLQICRT 540 2 TaPAO4-2A/1-490 446 SG----MEAAEECRRRLLMSKGVPDLVQVAGA-ACEEMADVVAPLQICRT 490 bits Motif 4 V T H H LS R EQ N Y V T Y T KY S N I E S A OsPAO4/1-487 445 SG----IAAADECRKRILMQKGIPDLVQVK---AYEEMAGVIAPLQICRT 487 CS D A V F SA ANARHT H FY TV 0 L A S 1 2 3 4 5 6 7 8 9 G F GE 10 11 12 13 14 G15 16 V17 H18 G19 A20 21 22 23 24 TaPAO5-2B/1-526 481 SG----IDAAEDCRRRLSTQLGISDLFQVGKIVMREEMAEVMVPLQISRL 526 TaPAO5-2D/1-492 447 SG----IDAAEDCRRRLSTQLGISDLFQVGKIVMREEMTEVMVPLQISRL 492 TaPAO5-2A/1-487 442 SG----IGAAEDCRRRLSTQLGISDLFQVGKIVMREEMTEVMVPLQISRL 487 4 OsPAO5/1-492 447 SG----IVAAEDCRRHLSTQLGISDLFQVGKIIMREEMTEVMVPFQISRL 492 2 Motif 5 bits D M I LAMQR L KPT P VP VADY AY Y FAEP R TaPAO1-3A/1-510 497 SG----VREADQLLQHYR------510 S YT D V P D S S M A Q VM N F I FKH M S Q GQ TNG G L N R Y PL LR I L V EE A V K S F S PT QC F M K TI A TDPP PDQFQRDAVSAAG G I K F I L AV T P P A N C A Y KA TaPAO1-3B/1-507 494 SG----VREADRLLQHYR------507 0 C YF A H L GR ED K SD 1 2 3 4 5 6 7 8 9

G 38 39 40 41 10 L11 12 13 H14 15 16 17 18 19 20 21 22 V23 24 M25 26 27 28 29 30 31 32 33 E34 35 36 37 TaPAO1-3D/1-491 478 SG----VREADRLLQHYR------491 OsPAO1/1-512 492 SG----VREANRLLQHYRGGANHTT------512 TaPAO8-1A/1-585 562 SG----AIVANTLVSVAQHSELLDAVGI------585 4 TaPAOUn/1-585 562 SG----AIVANTLVSVAQHSELLDAVGI------585 2 M Motif 6 bits AG R I V A S S KR SN GFT TaPAO8-5D/1-582 559 SG----AIVANTLVSVAQHSELLDAVGI------582 AAS RA D KR P I E I T P I I AGL A NS E YA Q S R K E E TA A C QV R CE G V V P V A K H G S S L A Q D N I A GG K S P G K C TaPAO8-5B/1-585 562 SG----AIVANTLVSVAQHSELLDAVGI------585 0 DQRDHQ I VALV S FG LCSGTH WDLSRR 1 2 3 4 5 6 7 8 9 I 10 G11 12 G13 14 15 G16 17 18 19 20 21 22 L23 24 25 26 27 28 29 TaPAO9-2A/1-474 455 SG----RRAAEALAKDGGLSQSLS------474 TaPAO9-2B/1-471 452 SG----RRAVEALVKDGGLSQSLS------471 TaPAO2-2A/1-340 339 TY------340 10 8 stage TaPAO6−7D TaPAO6−7A TaPAO6−7B TaPAO11−7A TaPAO1−3A TaPAO9−2A TaPAO9−2B TaPAO7−4D TaPAO10−5A TaPAO1−3B TaPAO1−3D TaPAO2−2A TaPAO11−7B TaPAO7−4A TaPAO7−4B TaPAO10−4B TaPAO5−2A TaPAO5−2B TaPAO5−2D TaPAO8−1A TaPAO8−5D TaPAO3−2D TaPAO8−5B TaPAOUn TaPAO11−7D TaPAO3−2A TaPAO3−2B TaPAO4−2B TaPAO4−2A TaPAO4−2D

6 ripening ﬂag_leaf_blade grain

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grain soft_dough grain

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lemma 2 glumes

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The copyright holder for this preprint this for holder copyright The ﬂag_leaf_sheath

ﬁfth_leaf_blade . anthesis ﬂag_leaf ovary anthers lemma glumes

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3_lea root third_leaf_sheath doi: doi: third_leaf_blade shoot_apical_meristem root ﬁrst_leaf_blade

seedling ﬁrst_leaf_sheath stem_axis coleoptile radicle bioRxiv preprint preprint bioRxiv (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is It is in perpetuity. preprint the to display a license bioRxiv granted has who the author/funder, is review) peer by certified not was (which bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.189209; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

10 10 10 control control A (SRP043554, Manitou) *** B (SRP060670, Chinese Spring) C (SRP078208, Chara) control 9 cold 9 fusarium 9 fusarium 8 8 8 * *** ***

7 *** 7 7 ***

6 6 6 *** *** * *** *** *** *** * *** * *

5 ** 5 * 5 *** * 2 *** ** ***

4 4 *** 4 *** Log (TPM+1) ** * * 3 3 *** 3 ** ** *** ** 2 2 **

*** 2 *

1 1 * 1 * * * ** 0 0 0

10 10 10 D (SRP045409, TAM 107) control E (SRP045409, TAM 107) control F (SRP045409, TAM 107) control 9 drought & heat 9 heat 9 drought ** 8 ** 8 8

7 7 7 *

6 6 * 6 * * * 5 5 5 2 * * 4 4 *** 4 * Log (TPM+1) *

3 3 3 ** ** ** * * * *** *** 2 2 2 *** * * 1 * 1 1 * * * * 0 0 0

10 10 10 G (SRP068165, Giza 168) control H (SRP068165, Gemmiza 10) control I (ERP009837, Riband) control 9 PEG 9 PEG 9 Zymoseptoria 8 8 8

7 *** 7 7

6 6 6 ** * **

5 5 *** 5 *** ** 2 *

4 4 *** 4 * ** *** ** Log (TPM+1) *** ***

3 *** 3 3 *** *** ** * ** * *** ***

2 *** 2 2 **

1 1 1 * 0 0 0

10 10 10 control control control 9 J (DRP000768, Chinese Spring) * 9 K (SRP041017, N9134) 9 L (SRP041017, N9134) -P powd._mildew. stripe_rust 8 8 8 *

7 7 7

6 6 6 * * * 5 5 5 * 2

4 4 * 4 * ** Log (TPM+1) 3 * 3 3 *** 2 2 2 * 1 1 1 ** 0 0 0 AO8-5B TaPAOUn TaPAOUn TaPAOUn TaPAO2-2A TaPAO3-2A TaPAO3-2B TaPAO4-2A TaPAO4-2B TaPAO5-2A TaPAO5-2B TaPAO6-7A TaPAO6-7B TaPAO9-2A TaPAO9-2B TaPAO1-3A TaPAO1-3B TaPAO7-4A TaPAO7-4B TaPAO8-1A TaP TaPAO8-5D TaPAO3-2D TaPAO4-2D TaPAO5-2D TaPAO6-7D TaPAO1-3D TaPAO7-4D TaPAO8-1A TaPAO8-5B TaPAO2-2A TaPAO3-2A TaPAO3-2B TaPAO4-2A TaPAO4-2B TaPAO5-2A TaPAO5-2B TaPAO6-7A TaPAO6-7B TaPAO9-2A TaPAO9-2B TaPAO1-3A TaPAO1-3B TaPAO7-4A TaPAO7-4B TaPAO8-1A TaPAO8-5B TaPAO2-2A TaPAO3-2A TaPAO3-2B TaPAO4-2A TaPAO4-2B TaPAO5-2A TaPAO5-2B TaPAO6-7A TaPAO6-7B TaPAO9-2A TaPAO9-2B TaPAO1-3A TaPAO1-3B TaPAO7-4A TaPAO7-4B TaPAO8-5D TaPAO3-2D TaPAO4-2D TaPAO5-2D TaPAO6-7D TaPAO1-3D TaPAO7-4D TaPAO8-5D TaPAO3-2D TaPAO4-2D TaPAO5-2D TaPAO6-7D TaPAO1-3D TaPAO7-4D TaPAO10-4B TaPAO10-5A TaPAO11-7A TaPAO11-7B TaPAO11-7D TaPAO10-4B TaPAO10-5A TaPAO11-7A TaPAO11-7B TaPAO10-4B TaPAO10-5A TaPAO11-7A TaPAO11-7B TaPAO11-7D TaPAO11-7D

M B control N B control O B control TaPAO1 100 cold 100 fusarium 100 fusarium TaPAO2 B-dominant TaPAO3 expression (TPM) expression (TPM) expression (TPM) TaPAO4 D-suppressed 0 20 0 20 20 TaPAO5 0 TaPAO6 80 80 80 30 200 100 TaPAO7 400 60 TaPAO8 200 TaPAO9 40 600 40 40 90 TaPAO10 60 60 300 60 TaPAO11 B B B A 800 A A

60 60 60 40 40 40

80 80 80 20 20 20

100 100 100

A 20 40 60 80 D A 20 40 60 80 D A 20 40 60 80 D 100 100 100

SRP043554 D Manitou SRP060670 D Chinese Spring (Dt7EL) SRP078208 D Chara

Figure 7 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.189209; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A B TaPAO5-2D 3 1 20 15 10 15 5 0 10 FPKM FPKM 4 2 15 5 10 5 0 0 C04 C23 C04 C23 C04 C23

condition condition C 0 1.3 2.7 4.1 5.5 6.9 8.2 9.6 11.0 12.4 0 1.3 2.7 4.1 5.5 6.9 8.2 9.6 11.0 12.4

mean expression, by transcript (FPKM) mean expression, by transcript (FPKM)

4 4

3 3

2 2

1 1

TaPAO5-2D: 4°C TaPAO5-2D: 23°C

624662000 624664000 624666000 624662000 624664000 624666000 genomic position genomic position

Figure 8 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.189209; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Supplementary File 1

>sp|Q0J290|PAO7_ORYSJ Polyamine oxidase 7 OS=Oryza sativa subsp. japonica OX=39947 GN=PAO7 PE=1 SV=1 MTKPTTMAIFLSIVLLSMAQLPSLVAGTGRPRVIIIGAGISGISAGKRLSEAGITDILIL EATDHIGGRMHKQRFAGVNVEIGANWVEGVNGEKMNPIWPIVNSTLKLRNFLSDFDSLAQ NVYKDGGLCDAAYVQKRIDLADEADKSGENLSATLHPSGRDDMSILSMQRLNNHLPNGPS SPVDMVVDYFTYDYEFAEPPRVTSLRNTVPLPTFTDFGDDNYFVADQRGYEAVVYYLAGQ YLEADKSGNIVDARLQLNKVVREISYSSTGVTVKTEDNSTYQADYVMVSASLGVLQSDLI QFKPQLPSWKILAIYQFDMAVYTKIFVKFPKKFWPEGAGREFFLYASTRRGYYGVWQEFE KQYPDANVLLVTVTDEESRRIEQQPDSQTKAEIMEVVRSMFPDEDVPDATDILVPRWWSD RFFQGSFSNWPIGVSRYEHDQLRAPVGRVYFTGEHTSERYNGYVHGAYLAGIYA >sp|Q9SU79|PAO5_ARATH Probable polyamine oxidase 5 OS=Arabidopsis thaliana OX=3702 GN=PAO5 PE=1 SV=1 MAKKARIVIIGAGMAGLTAANKLYTSSNNTFELSVVEGGSRIGGRINTSEFSSEKIEMGA TWIHGIGGSPVYRIAKETGSLVSDEPWECMDSTIDKAKTFAEGGFEIEPSIVESISGLFT ALMELAQGKEISQSDADLSRLAHIYETATRVCSKGSSTSVGSFLKSGFDAYWDSISNGGE EGVKGYGKWSRKSLEEAIFTMFSNTQRTYTSADELSTLDFAAESEYQMFPGEEITIAKGY LSVIHHLASVLPQGVIQLNRKVTKIEWQSNEVKLHFSDGSVVFADHVIVTVSLGVLKAGI ETDAELFSPPLPDFKSDAIRRLGYGVVNKLFVEMSQRKFPSLQLVFDREDSEFRFVKIPW WMRRTATITPIHSNSKVLLSWFAGKEALELEKLTDEEIKDAVMTTISCLTGKEVKNDTAK PLTNGSLNDDDEAMKITKVLKSKWGSDPLFRGSYSYVAVGSSGDDLDAMAEPLPKINKKV GQVNGHDQAKVHELQVMFAGEATHRTHYSTTHGAYYSGLREANRLLKHYKCNF >sp|Q9LYT1|PAO3_ARATH Polyamine oxidase 3 OS=Arabidopsis thaliana OX=3702 GN=PAO3 PE=1 SV=1 MESGGKTNRQLRKAICVSTDEKMKKKRSPSVIVIGGGMAGISAARTLQDASFQVVVLESR DRIGGRVHTDYSFGFPVDLGASWLHGVCKENPLAAVIGRLGLPLYRTSGDNSVLYDHDLE SYALFDKAGNQVSQELVTKVGENFEHILEEICKVRDEQDEDMSIAQAFSIVFKRNPELRL EGLAHNVLQWYLCRMEGWFAADAETISAKCWDQEELLPGGHGLMVRGYRPVINTLSKGLD IRLSHRITKISRRYSGVKVTTEKGDTFVADAAVIALPLGVLKSGMITFEPKLPQWKQEAI NDLGVGIENKIILNFDNVFWPNVEFLGVVAETSYGCSYFLNLHKATSHPVLVYMPAGQLA RDIEKKSDEAAANFAFSQLQKILPDASSPINYLVSRWGSDINSLGSYSYDIVNKPHDLYE RLRVPLDNLFFAGEATSSSYPGSVHGAYSTGVLAAEDCRMRVLERYGELEHEMEEEAPAS VPLLISRM >sp|Q7X809|PAO3_ORYSJ Polyamine oxidase 3 OS=Oryza sativa subsp. japonica OX=39947 GN=PAO3 PE=1 SV=2 MANNSSYGENVRRKSHTPSAIVIGSGFAGIAAANALRNASFEVVLLESRDRIGGRIHTDY SFGFPVDLGASWLHGVCEENPLAPIIGRLGLPLYRTSGDDSVLFDHDLESYALYDTKGHQ VPQELVEKIGKVFETILEETGKLREETKEDISIAKAIAIVMERNPHLRQEGIAHDVLQWY LCRMEGWFATDADAISLQGWDQEVLLPGGHGLMVRGYRPVINTLAKGLDIRLGHRVVEIV RHRNRVEVTVSSGKTFVADAAVIAVPLGVLKANTIKFEPRLPEWKEEAIRELSVGVENKI ILHFSEVFWPNVEFLGVVSSTTYGCSYFLNLHKATGHPVLVYMPAGRLACDIEKLSDEAA AQFAFSQLKKILPNAAEPIHYLVSHWGSDENTLGSYTFDGVGKPRDLYEKLRIPVDNLFF AGEATSVQYTGTVHGAFSTGLMAAEECRMRVLERFRELDMLEMCHPAMGEQTATVSVPLL ISRL >sp|Q5NAI7|PAO1_ORYSJ Polyamine oxidase 1 OS=Oryza sativa subsp. japonica OX=39947 GN=PAO1 PE=1 SV=1 MVAKKPRVVVVGAGISGLAAAHRLCGAGGDRFEVAVVEAGDRVGGRILTSEFAGHRVEMG ATWVQGVVGSPVYALARDAGALGEEEGRGLPYERMDGFPDRVLTVAEGGEVVDADTVAGP IEELYRGMMEAARAGEAGGGGGVEEYLRRGLRAYQAARSAGGGGGGGKELEEVDEALLAM HINRERTDTSADDLGDLDLTAEGEYRDFPGEHVTIPGGYSRVVERLAAALPPGTVRLGLR LRRLKWGGTPVRLHFADGAPPLTADHVILTVSLGVLKASLGNKDTAGVGAAAIAFDPPLP PFKREAVARLGFGVVNKLFMEVEAVAPSEPEDVAGVQPAAAGFPFLHMAFRGHVSKIPWW MRGTESICPVHAGSTVALAWFAGREAAHLESLPDDDVIRGAHATLDSFLPAAPRWRVRRI KRSGWATDPLFLGSYSYVAVGSSGDDLDRMAEPLPRGPDAAADERPPSPRLLFAGEATHR THYSTTHAAYLSGVREANRLLQHYRGGANHTT >sp|Q0J954|PAO5_ORYSJ Polyamine oxidase 5 OS=Oryza sativa subsp. japonica OX=39947 GN=PAO5 PE=1 SV=1 MDQPSNGFAAGGLFLRHIDGQNASPPSVIVIGGGISGIAAARALSNASFKVTLLESRDRL GGRVHTDYSFGCPIDMGASWLHGVCNENSLAPLIRLLGLRLYRTSGDNSVLYDHDLESYA LFDKDGRQVPQEIVTKVGETFEKILKETVKVRAEHEDDMPLIQAISIVLDRNPHLKLDGL QYEVLQWCICRLEAWFATDVDNISLKNWDQEHVLTGGHGLMVHGYDPVIKALAQDLDIHL NHRVTKIIQRYNKTIVCVEDGTSFVADAAIITVPLGVLKANIIKFEPELPDWKLSSISDL GIGIENKIALRFNSVFWPNVEVLGRVAPTSNACGYFLNLHKATGHPVLVCMVAGRFAYEF EKLSDEESVNFVMSQLKKMLPGATEPVQYLVSRWGTDPNSLGSYSCDLVGKPADLYERFC APVGNLFFAGEAACIDHSGSVHGAYSSGIVAAEDCRRHLSTQLGISDLFQVGKIIMREEM TEVMVPFQISRL >sp|Q7XR46|PAO4_ORYSJ Polyamine oxidase 4 OS=Oryza sativa subsp. japonica OX=39947 GN=PAO4 PE=1 SV=1 MDPNSLKTGGLLLPTIERQCASPPSVIVIGGGISGVAAARALSNASFEVTVLESRDRVGG RVHTDYSFGCPIDMGASWLHGVCNENSLAPLIGYLGLKLYRTSGDNSVLYDHDLESYALF DKAGHQVSKETVAKVEETFERILDETVKVRDEQEHDMPLLQAISLVLERHPHLKLQGIDD QVLQWCVCRLEAWFAADADEISLKNWDQEHVLTGGHGLMVNGYYPIIQALAQGLDIRLNQ RVTKIARQFNGVTVTTEDGTSYSADACIITVPLGVLKANIIKFEPELPSWKSSAIADLGV GIENKIAMHFDTVFWPNVEVLGMVGPTPKACGYFLNLHKATGNPVLVYMAAGRFAQEVEK bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.189209; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

LSDKEAVDLVMSHLKKMLPDATEPTKYLVSRWGSDPNSLGSYSCDLVGKPADVSARFAAP VENLYFAGEAASADHSGSVHGAYSSGIAAADECRKRILMQKGIPDLVQVKAYEEMAGVIA PLQICRT >sp|Q9SKX5|PAO2_ARATH Polyamine oxidase 2 OS=Arabidopsis thaliana OX=3702 GN=PAO2 PE=1 SV=1 MESRKNSDRQMRRANCFSAGERMKTRSPSVIVIGGGFGGISAARTLQDASFQVMVLESRD RIGGRVHTDYSFGFPVDLGASWLHGVCKENPLAPVIGRLGLPLYRTSGDNSVLYDHDLES YALFDMDGNQVPQELVTQIGVTFERILEEINKVRDEQDADISISQAFSIVFSRKPELRLE GLAHNVLQWYVCRMEGWFAADAETISAKCWDQEELLPGGHGLMVRGYRPVINTLAKGLDI RVGHRVTKIVRRYNGVKVTTENGQTFVADAAVIAVPLGVLKSGTIKFEPKLPEWKQEAIN DLGVGIENKIILHFEKVFWPKVEFLGVVAETSYGCSYFLNLHKATGHPVLVYMPAGQLAK DIEKMSDEAAANFAVLQLQRILPDALPPVQYLVSRWGSDVNSMGSYSYDIVGKPHDLYER LRVPVDNLFFAGEATSSSFPGSVHGAYSTGLMAAEDCRMRVLERYGELDLFQPVMGEEGP ASVPLLISRL >sp|A0A0P0XM10|PAO6_ORYSJ Polyamine oxidase 6 OS=Oryza sativa subsp. japonica OX=39947 GN=PAO6 PE=2 SV=1 MTKPTTMAIFLVLALSIAQLLPSLVAGTGRPRVIIVGAGISGISAGKRIWEAGIADVLIL EATDRIGGRMHKQSFAGVNVEIGANWVEGVNGEKKNPIWPIVNSTLKLRSFRSDFDSLAQ NVYKDGGLCDEAYVQKRMDRADEVDKSGENLSATLHPSGRDDMSILSMQRLNDHLPNGPS SPVDMAVDYFTYDYEFAEPPRVTSLQNTVPLPTFTDFGDDTYFVADQRGYESVVHHLAGQ YLNADKSGNIADARLKLNKVVREISYSSTGVTVKTEDNSTYQADYVMVSASLGVLQSDLI QFKPQLPSWKILAIYQFDMAVYTKIFVKFPKKFWPEGAGREFFLYASTRRGYYGVWQEFE KQYPDANVLLVTVTDEESRRIEQQPDSQTKAEIMEVVRCMFPDEDVPDATDILVPRWWSD RFFRGSFSNWPIGVSRYEYDQLRAPVGRVYFTGEHTSERYNGYVHGAYLAGIDSAEILIN CAQKKMCKYNVGGKHG >sp|Q8H191|PAO4_ARATH Probable polyamine oxidase 4 OS=Arabidopsis thaliana OX=3702 GN=PAO4 PE=1 SV=1 MDKKKNSFPDNLPEGTISELMQKQNNVQPSVIVIGSGISGLAAARNLSEASFKVTVLESR DRIGGRIHTDYSFGCPVDMGASWLHGVSDENPLAPIIRRLGLTLYRTSGDDSILYDHDLE SYGLFDMHGNKIPPQLVTKVGDAFKRILEETEKIRDETANDMSVLQGISIVLDRNPELRQ EGMAYEVLQWYLCRMEAWFAVDANLISLKCWDQDECLSGGHGLMVQGYEPVIRTIAKDLD IRLNHRVTKVVRTSNNKVIVAVEGGTNFVADAVIITVPIGVLKANLIQFEPELPQWKTSA ISGLGVGNENKIALRFDRAFWPNVEFLGMVAPTSYACGYFLNLHKATGHPVLVYMAAGNL AQDLEKLSDEATANFVMLQLKKMFPDAPDPAQYLVTRWGTDPNTLGCYAYDVVGMPEDLY PRLGEPVDNIFFGGEAVNVEHQGSAHGAFLAGVSASQNCQRYIFERLGAWEKLKLVSLMG NSDILETATVPLQISRM >sp|Q9FNA2|PAO1_ARATH Polyamine oxidase 1 OS=Arabidopsis thaliana OX=3702 GN=PAO1 PE=1 SV=1 MSTASVIIIGAGISGISAAKVLVENGVEDVLILEATDRIGGRIHKQNFGDVPVELGAGWI AGVGGKESNPVWELASRFNLRTCFSDYTNARFNIYDRSGKIFPTGIASDSYKKAVDSAIL KLKSLEAQCSGQVAEEAPSSPKTPIELAIDFILHDFEMAEVEPISTYVDFGEREFLVADE RGYECLLYKMAEEFLVTSHGNILDYRLKLNQVVREVQQSRNGVVVKTEDGSVYEANYVIV SASIGVLQSDLLSFQPLLPRWKTEAIQKCDVMVYTKIFLKFPQCFWPCGPGQEFFIYAHE QRGYFTFWQHMENAYPGSNILVVTLTNEQSKRVEAQSDQETMKEAMSVLRDMFGATIPYA TDILVPRWWNNRFQRGSYSNYPMISDNQLLQNIKAPVGRIFFTGEHTSEKFSGYVHGGYL AGIDTSKSLLEEMKQSLLLQPLLAFTESLTLTHQKPNNSQIYTNVKFISGTS bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.189209; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A B control B B control C B control TaPAO1 100 heat 100 drought 100 drought & heat TaPAO2 TaPAO3 expression (TPM) expression (TPM) TaPAO4 expression (TPM) 20 0 20 20 TaPAO5 0 80 80 80 0 TaPAO6 50 TaPAO7 50 50 TaPAO8 100 100 100 TaPAO9 40 150 40 40 TaPAO10 60 60 150 60 150 B B B TaPAO11 A 200 A A 200 200

60 60 60 40 40 40

80 80 80 20 20

100 100 100

A 20 40 60 80 D A 20 40 60 80 D A 20 40 60 80 D 100 100 100

SRP045409 D TAM 107 SRP045409 D TAM 107 SRP045409 D TAM 107 D B control E B control F B control 100 PEG 100 PEG 100 Zymoseptoria expression (TPM) expression (TPM) expression (TPM) 0 20 20 0 20 0 80 30 80 50 80 25 60 100 50

40 90 40 150 40 75 60 60 60 B B B 100 A A 200 A

125 60 60 60 40 40 40

80 80 80 20 20 20

100 100 100 125

A 20 40 60 80 D A 20 40 60 80 D A 20 40 60 80 D 100 100 100

SRP068165 D Giza 168 (tolerant) SRP068165 D Gemmiza 10 (sensetive) ERP009837 D Riband G B control H B control I B control 100 -P 100 powdery_mildew 100 stripe_rust

expression (TPM) expression (TPM) expression (TPM) 0 20 0 20 20 0 80 80 80 100 50 50

200 100 100 40 300 40 40 60 60 60 B B B A 400 A A

60 60 60 40 40 40

80 80 80 20 20 20

100 100 100

A 20 40 60 80 D A 20 40 60 80 D A 20 40 60 80 D 100 100 100 DRP000768 D Chinese Spring SRP041017 D N9134 SRP041017 D N9134

Supplementary Fig. S1. Ternary plot showing relative expression abundance of TaPAO genes under different stress conditions. In each ternary plot, a circle or a triangles reflects the relative contribution of homoeologs of a gene under normal or stress condition, respectively and their sizes indicate the total expression in TPM. The data code for each study and the evaluated wheat cultivar are also indicated at bottom of subfigures. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.189209; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

TraesCS2A02G548100 (TaPAO5-2A): 23°C TraesCS2A02G548100 (TaPAO5-2A): 4°C TaPAO8-1A: 23°C TaPAO8-1A: 4°C 0 0.98 1.97 3.05 4.04 5.12 6.1 7.19 8.17 9.25 10.2411.32 12.3 13.3914.3715.4516.4417.5218.5119.59 0 0.98 1.97 3.05 4.04 5.12 6.1 7.19 8.17 9.25 10.2411.32 12.3 13.3914.3715.4516.4417.5218.5119.59 0 0.72 1.47 2.22 2.98 3.73 4.48 5.24 5.99 6.75 0 0.72 1.47 2.22 2.98 3.73 4.48 5.24 5.99 6.75 mean expression, by transcript mean expression, by transcript mean expression, by transcript mean expression, by transcript

755927000 755929000 755931000 755927000 755929000 755931000 570132000 570136000 570140000 570132000 570136000 570140000

TraesCS2B02G490100 (TaPAO3-2B): 23°C TraesCS2B02G490100 (TaPAO3-2B): 4°C TaPAO4-2A: 23°C TaPAO4-2A: 4°C 0 0.85 1.7 2.64 3.49 4.42 5.27 6.21 7.06 7.99 8.85 9.78 10.6311.5712.4213.35 14.2 15.1415.9916.93 0 0.85 1.7 2.64 3.49 4.42 5.27 6.21 7.06 7.99 8.85 9.78 10.6311.5712.4213.35 14.2 15.1415.9916.93 0 9.37 19.21 29.05 38.9 48.74 58.58 68.42 78.26 88.1 0 9.37 19.21 29.05 38.9 48.74 58.58 68.42 78.26 88.1

mean expression, by transcript mean expression, by transcript mean expression, by transcript mean expression, by transcript

687469000 687471000 687473000 687469000 687471000 687473000 755974000 755975000 755976000 755974000 755975000 755976000

TraesCS2B02G579000 (TaPAO5-2B): 23°C TraesCS2B02G579000 (TaPAO5-2B): 4°C TaPAO11-7D: 23°C TaPAO11-7D: 4°C

0 1.19 2.39 3.7 4.9 6.21 7.41 8.72 9.91 11.2312.4213.7414.9316.2417.4418.7519.9521.2622.4623.77 0 1.19 2.39 3.7 4.9 6.21 7.41 8.72 9.91 11.2312.4213.7414.9316.2417.4418.7519.9521.2622.4623.77 0 48 98 149 200 250 301 351 402 453 0 48 98 149 200 250 301 351 402 453

mean expression, by transcript mean expression, by transcript mean expression, by transcript mean expression, by transcript

767128000 767130000 767132000 767128000 767130000 767132000 486057000 486059000 486061000 486057000 486059000 486061000 genomic position genomic position TraesCS5B02G529400 (TaPAO8-5B)4: 23°C TraesCS5B02G529400 (TaPAO8-5B): 4°C TraesCS5D02G528500 (TaPAO85D): 23°C TraesCS5D02G528500 (TaPAO8-5D): 4°C 0 0.56 1.12 1.73 2.29 2.9 3.46 4.07 4.63 5.24 5.8 6.42 6.97 7.59 8.14 8.76 9.32 9.93 10.49 11.1 0 0.56 1.12 1.73 2.29 2.9 3.46 4.07 4.63 5.24 5.8 6.42 6.97 7.59 8.14 8.76 9.32 9.93 10.49 11.1 0 0.82 1.65 2.55 3.38 4.29 5.11 6.02 6.84 7.75 8.57 9.48 10.3 11.2112.0312.9413.7614.6715.49 16.4 0 0.82 1.65 2.55 3.38 4.29 5.11 6.02 6.84 7.75 8.57 9.48 10.3 11.2112.0312.9413.7614.6715.49 16.4 mean expression, by transcript mean expression, by transcript

mean expression, by transcript mean expression, by transcript

687804000 687808000 687812000 687804000 687808000 687812000 545216000 545220000 545224000 545216000 545220000 545224000 genomic position genomic position

Supplementary Fig. S2 Structure and expression levels in FPKM of distinct isoforms of eight TaPAO genes in normal (23°C) and stress (4°C) from SRP043554 experiment. Expression levels are shown in varying shades of yellow.