Genome wide identification, evolution and expression analysis of the aspartic protease gene family during rapid growth of moso bamboo (phyllostachys edulis) shoots

RESEARCH ARTICLE Open Access Genome wide identification, evolution and expression analysis of the aspartic protease gene family during rapid growth of moso bamboo (Phyllostachys edulis) shoots Xiaqin[.]

R E S E A R C H A R T I C L E Open Access

Genome-wide identification, evolution and

expression analysis of the aspartic protease

gene family during rapid growth of moso

bamboo (Phyllostachys edulis) shoots

Xiaqin Wang1,2,3, Xinyang Yan1,2, Shubin Li1, Yun Jing1, Lianfeng Gu1, Shuangquan Zou1,2, Jin Zhang3*and Bobin Liu1,2*

Abstract

Background: Aspartic proteases (APs) are a class of aspartic peptidases belonging to nine proteolytic enzyme families whose members are widely distributed in biological organisms APs play essential functions during plant development and environmental adaptation However, there are few reports about APs in fast-growing moso bamboo

Result: In this study, we identified a total of 129 AP proteins (PhAPs) encoded by the moso bamboo genome

Phylogenetic and gene structure analyses showed that these 129 PhAPs could be divided into three categories

(categories A, B and C) The PhAP gene family in moso bamboo may have undergone gene expansion, especially the members of categories A and B, although homologs of some members in category C have been lost The

chromosomal location of PhAPs suggested that segmental and tandem duplication events were critical for PhAP gene expansion Promoter analysis revealed that PhAPs in moso bamboo may be involved in plant development and

responses to environmental stress Furthermore, PhAPs showed tissue-specific expression patterns and may play important roles in rapid growth, including programmed cell death, cell division and elongation, by integrating

environmental signals such as light and gibberellin signals

Conclusion: Comprehensive analysis of the AP gene family in moso bamboo suggests that PhAPs have experienced gene expansion that is distinct from that in rice and may play an important role in moso bamboo organ development and rapid growth Our results provide a direction and lay a foundation for further analysis of plant AP genes to clarify their function during rapid growth

Keywords: Aspartic protease, Moso bamboo, Programmed cell death, Rapid growth

© The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/ ) applies to the

* Correspondence: zhangj@zafu.edu.cn ; liubobin@fafu.edu.cn

3

State Key Laboratory of Subtropical Silviculture, School of Forestry and

Biotechnology, Zhejiang A&F University, Zhejiang 311300, Hangzhou, China

1 College of Forestry, Fujian Agriculture and Forestry University, Fuzhou

350002, China

Full list of author information is available at the end of the article

Aspartic proteinases (APs; Enzyme Commission 3.4.23)

are proteolytic enzymes and play important roles in

APs have two conserved motifs with catalytic activity: an

Asp-Thr-Gly (DTG) motif and an Asp-Ser-Gly (DSG)

sub-families based on their tertiary structure and

important biological processes that are involved in

devel-opment, nutrition, pathogenesis, disease and so on and

Most plant APs are grouped into the A1 family and

exhibit the two basic features of A1 family members:

one features is that they are active under acidic

condi-tions, and the other is that their catalytic activity can be

1980s, plant APs have been purified via pepstatin

A-agarose columns and detected in various plant species

[3,4,10] Plant APs can be classified into three

categor-ies: typical APs, nucellin-like APs and atypical APs [1,

9] Typical APs contain a plant-specific insert (PSI)

simi-lar to that of saposin-like proteins, but it is removed

similar to nucellins in barley ovules [11] The

character-istics of atypical APs are intermediate between those of

has been detected in immature, mature, and germinated

seeds in wheat, and the expression pattern showed a role

APs are also considered to be responsible for protein

processing and degradation, such as plant senescence,

programmed cell death (PCD), reproduction, and stress

devel-opment With the development of DNA sequencing

technology, members of plant AP gene families have

and poplar [22], revealing gene expansion and functional

diversity [12,22]

The function of plant APs has been determined

pri-marily in seeds, including dormant seeds and different

dur-ing seed development, plant APs are involved in seed

storage protein processing on the basis of the 2S

albu-min process in vitro and colocalization with proteins in

the plant body [25] During seed germination, plant APs

are considered to be involved in seed storage protein

PROTEASE IN GUARD CELL 1 (ASPG1) was reported

to promote seed germination by accelerating the

break-down of seed storage proteins [28] In addition to their

involvement in seed development and germination, APs

participate in the degradation of insect proteins, allowing

carnivorous plants to obtain nitrogen from those sources [15,29] Plant APs also play roles in the response to bi-otic and abibi-otic stresses ASPG1 is abscisic acid (ABA) inducible, and Arabidopsis plants overexpressing this gene had in increased ability to resist drought stress because of the participation of the transgene in ABA-dependent

(CDR1), an atypical plant aspartic proteinase, exhibits the ability to induce systemic defense responses against

protease gene, can afford powdery mildew resistance but reduces Botrytis cinerea resistance by regulating the sali-cylic acid and MeJA signaling pathways [19] Plant APs also play roles in plant development, such as reproduction and lateral root formation OsAP65 has been proposed to

be involved in biosynthesis of compounds that are essen-tial to pollen germination and pollen tube growth in rice

been speculated to participate in gametogenesis and pollen guidance [18] Recently, an atypical aspartic prote-ase, Atypical Aspartic Protease in Roots 1 (ASPR1), was de-termined to suppress primary root growth and lateral root development [33] Altogether, plant APs are important proteins that are involved in various aspects of plant de-velopment and responses to environmental changes Some plant APs also play an important role in regulat-ing PCD In barley, a gene encodregulat-ing an aspartic protease-like protein (‘nucellin’) was highly expressed after pollination, which was synchronized to nuclear cell degeneration characteristic of PCD [11] Phytepsin, a vacuolar aspartic proteinase that is a plant homolog of cathepsin D and mediates PCD in barley, is highly expressed during the active autolysis of the root cap and

in tracheary elements and sieve cells [34] In rice, the transcripts of OsAP25 and OsAP37 in anthers are acti-vated by ETERNAL TAPETUM 1 (EAT1) to regulate

OF CELL SURVIVAL 1 (PCS1) encodes an aspartic pro-tease, and compared with wild type, loss-of-function mutants experience gametophyte degeneration and cell death of developing embryos [36] AP proteins have also been identified in the plant cell wall, and cis-elements related to secondary cell wall (SCW) thickening and PCD, such as SNBE, TERE, and SMRE, were discovered upstream of partial AP genes from poplar, strongly sug-gesting that APs play important roles in SCW and PCD [37–41] To date, there are many reports on plant AP function in model plant species such as Arabidopsis and rice However, the function of APs in rapid-growing plant species such as bamboo is still unclear

Bamboo is a member of the Gramineae family, is widely distributed worldwide and is a rapid-growing plant species Bamboo forests can provide young

bamboo shoots for food, fibrous raw material, building

materials, raw materials for furniture and crafts and so on

within a short time [42] In addition to its economic

bene-fits, bamboo also has important ecological functions, such

as the ability to restore degraded landscapes and combat

(Phyl-lostachys edulis) planting area is approximately 3.27

mil-lion ha and constitutes most of the bamboo forest region

in China [43] Rapid growth of moso bamboo occurs after

the young bamboo shoots are covered with a shell and

emerge from the ground PCD was revealed to occur in

pith cavity formation during rapid bamboo growth [44]

During the bamboo rapid-growth stage, cell division

grad-ually decreases, while cell elongation and secondary cell

SCW formation are important biological events during

rapid growth of moso bamboo Members of the NAC,

MYB and LAC gene families have been identified as being

gene family has specifically been reported to be involved

in environmental responses [49] In addition to rapid

syn-thase [52] have also been widely studied in bamboo

Re-cently, a chromosome-level de novo genome assembly of

moso bamboo was provided, which, compared with the

previous version, was obviously improved in terms of the

assembly data and quality of the whole-genome

gen-omic data allows us to perform genome-wide gene

functional analyses in moso bamboo

Here, we identified a total of 129 PhAP proteins that

contain a conserved Asp domain from the moso bamboo

genome Phylogenetic analysis revealed that PhAP genes

might have experienced gene expansion via segmental

and tandem duplication Gene structure and motifs

indi-cated that the motifs of PhAPs were conserved, although

the gene structure has changed throughout evolutionary

history Expression pattern analysis showed that PhAPs

exhibited tissue-specific expression patterns, and several

sets of PhAPs may play important roles during moso

bamboo rapid growth Our study provides a strong

foun-dation for further research on the potential function of

these proteins in bamboo development and an improved

understanding of the AP gene family in fast-growing

nontimber forest species

Results

Genome-wide identification of AP genes from the moso

bamboo genome

After two rounds of moso bamboo genome searching via

HMMER v3 (the details of which are in the materials

and methods), a total of 129 Asp family proteins with a

conserved Asp domain were analyzed via the

these Asp proteins, 102 had two catalytic sequence motifs, Asp-Thr-Gly (DTG) and Asp-Ser-Gly (DSG), which are typical features of aspartic proteases; however, 18 proteins contained one catalytic motif, and nine proteins had no

named based on their relationships with homologous genes

in rice and are listed in Table S1 Other information on the members of the Asp gene family, including their chromo-somal localization, CDS, amino acid residue sequence, corresponding protein length, corresponding protein mo-lecular weight, and corresponding protein isoelectric point,

is also listed in Table S1

Phylogenetic relationships among the 129 moso bamboo Asp proteins were determined using an IQ-TREE procedure [54] The 129 moso bamboo Asp proteins fell into three dis-tinct categories (pink, blue and purple clades) and were termed categories A, B and C, respectively (Fig.1) From the predicted protein domain, we found that all PhAPs

were 16 moso bamboo category A PhAP members, eight of which contained signal peptides, and the Asp domain con-sisted of the Taxi_N and PSI domains (including SapB_1 and SapB_2) with two catalytic motifs (Fig.1and Table S1) However, there were no signal peptides or PSI domains and/or a lack or partial lack of catalytic motifs in the other

and C had 26 and 87 members, respectively, that contained the full-length Asp domain consisting of Taxi_N and Taxi_

C, except for PhAP7.4, PhAP31.3, PhAP7.2, PhAP87.1,

than half of the category B PhAPs are nucellin-like APs con-taining catalytic sites (Fig.1and Table S1), which is similar

to that which occurs rice [12] Category C, composed of atypical aspartic proteases, was the largest category (Fig.1) Most category B and C members contained a signal peptide, and it was notable that there were signal peptides and trans-membrane domains located in the N- and C-termini, re-spectively, of nine category B AP proteins (Fig.1)

Phylogenetic analysis of APs from moso bamboo and rice

To investigate the evolutionary relationship of the PhAP family, a phylogenetic tree was constructed using 129 PhAP and 92 OsAP full-length amino acid residue sequences

classed into three categories, as previously reported in Ara-bidopsis [9], rice [12], grape [21] and poplar [22] Category

A contained 16 PhAPs together with seven OsAPs; these proteins could be classified into seven subclades based on their relationships with their rice homologous proteins

genome encoded eight PhAP88 genes and only one homo-log in rice, which meant that AP88 underwent gene

B

A

PhAP22.2 PhAP53.1 PhAP91.1 PhAP70.2 PhAP68/76.1 PhAP8/21.1 PhAP8/21.3 PhAP48.1 PhAP57.1 PhAP57.2 PhAP57.3 PhAP57.5 PhAP20.2 PhAP58.1 PhAP56.1 PhAP73.1 PhAP58/73.1 PhAP66.2 PhAP17.1 PhAP25.1 PhAP14.1 PhAP7.2 PhAP7.3 PhAP45.2 PhAP87.1 PhAP4.2 PhAP39.2 PhAP16.1 PhAP16.3 PhAP71.1 PhAP87.2 PhAP93.2 PhAP93.4 PhAP93.6 PhAP50.3 PhAP50.1 PhAP27.1 PhAP28.1 PhAP72.1 PhAP72.3 PhAP36.1 PhAP61.1 PhAP5.1 PhAP43.2 PhAP12.3 PhAP12.1 PhAP40.2 PhAP65.1 PhAP15.2 PhAP19.2 PhAP35.1 PhAP74.2 PhAP31.2 PhAP59.3 PhAP59.1 PhAP7.4 PhAP31.3 PhAP23.1 PhAP52.1 PhAsp3.2 PhAsp1.1 PhAsp2.1 PhAP44/90.1 PhAP44/90.3 PhAP10.1 PhAP9.2 PhAP41.2 PhAP88.7 PhAP88.6 PhAP88.1 PhAP88.4

44 69 58

67 53 85

81

99100

63

92

98 90 100 99 100 100 94 60

100 100 97 78 100 100 100

71

10092

100100 100

68 64 65 100

65 74 37 100 100 100

49 57 60 56 54 26 39 100

49 57

9194 100 93

54 84 100 99 100 100

99100

74100 100 75 100

62 100 100 99 100

43

83 100

100100 100

81 57

97 97

99100 100

97100

94 98 100

84100 98 0

100 91 92 98 98 100

100 91 100 99 99 96 100 100

prot ein_dom ain

Asp SapB_1 TAXi_C TAXi_N signal pept ide

t ransm em brane dom ain PPR repeat

DYW_deam inase Met hylt ransf_29 ATP-synt _G Pect inest erase RRM_8 DXP_synt hase_n

Fig 1 (See legend on next page.)

were classified into category B, which could be further

di-vided into 13 subclades (Fig.2) Each subclade contained at

Cat-egory C contained 87 PhAPs and 70 OsAPs There was at

least one PhAP homolog, and PhAP57 and PhAP93

There were no homologous genes of OsAP77–87 in moso bamboo, indicating that the homologs in bamboo were lost

re-sults showed that the PhAP gene family in moso bamboo underwent specific evolutionary events after the divergence

of bamboo and rice

(See figure on previous page.)

Fig 1 Phylogenetic relationships and protein domain diagram of moso bamboo aspartic proteinases The left part shows the phylogenetic relationships of 129 APs from moso bamboo Categories A, B And C are shaded in pink, blue and purple, respectively The blue stars, red triangles and green circles represent APs containing 2, 1 and 0 catalytic sequences, respectively Bootstrap are shown close to the branch nodes The right part shows the protein domain, and the caption is shown in the upper left corner

B

A

C

OsAP31 PhAP31.2 PhAP31.1 OsAP74 PhAP74.2 PhAP74.1 OsAP59

PhAP31.

3 OsAP34 OsAP 33 Os AP35

PhAP

35.1 Os AP19 Ph AP1 9.2 Ph

AP19 .1 Os AP1 5 Ph AP1 5.2

Ph AP1 5.1

Os

AP65

Ph

AP65.

1

PhAP 65.

2

O sAP 23

Ph A P23.

2

Ph A P2 3.1

Os

AP5 2

Ph A P52.

2

Ph A P52.

1

O sAsp 3

PhA

sp3.

1

Ph As 2

O sAsp 2

Ph Asp 2.

2

Ph A

sp2 .1

Os A sp1

Ph Asp 1 1

Os A P4 0

Ph A P4 0 2

Ph AP 40 1

Os AP 4

O sAP 7

Os A 6

Ph A 6 /76.

1

Os A 70

Ph A 7 1

Ph A 70.

2

Os A 8

Os A 21

Ph A 8 /21.

1

Ph A 8/

1.

Ph A 8/

21.

Ph

OsAP

Ph A 48.

Os A 5

Ph

AP5 7

Ph A 5 7

Ph

AP5

7.2

PhA

P57.

6

PhA P5

7.3

Ph A

57.

4

Ph A 5 5 Os

AP2 0

Ph A 2 1

Ph

0.2 Os A 7 Ph

AP7

3.1

Os A 5

PhA P

8.2 Ph A

58.

1

Ph AP 5 /7 1

OsA

P5 Os

AP5 5 Os

AP5 6

Ph AP

5.3

PhA

1

5 2

OsA

P89 Os

AP2 2

PhA P22.

2

22.

1

OsAP 53

PhAP5

3.1

51

OsAP

46

OsAP 91

1.1

OsAP

66

PhAP

66.2

PhAP

66.1 OsAP 17

7.1

PhAP25.

2 PhAP25.

1 OsAP39 PhAP39.2 PhAP39.1 OsAP4 PhAP4.2 PhAP4OsAP14.1 PhAP14.2

OsAP7 PhAP7.2

OsAP12

PhAP 43.2

PhAP 43.1

Os AP11

Os AP18

PhA P18.1

Os AP16

PhAP 16.1

PhA P16.

2

PhA P16.

3

Os

AP67

OsAP 38

Os AP5

PhA P5.1

Ph AP 5.2

Os AP 37

Ph AP

37.1

Os A P62

Os A P60

O sAP 61

Ph

AP6 1 1

Os A P13

Ph A 1 1

Os A 7

Os A 9

Os A 6

Ph A 69.

1

Os A 7

P A 71.

2

Ph AP 7 1

Os A 3

Os A 29

Os A 3

Os A 2

Ph A 2 1

Os A 27

Ph A P2 7 1

Ph A 2 2

P hA P 3 2.

1

Os A P5 0

Ph A 5 1

Ph A P5 0 2

Ph A 5 3

Ph AP 3 2

Os AP

.2 PhA

sAP

Ph

P3

6.1

Ph A 3 2

Os A 6

Os A 6

Ph AP 6 1

Os

AP9 3

Ph A 93.

1

Ph A 9 2

PhA P

3.3

Ph A

93.

4

Ph

AP9

3.5

.6

Ph A

1

Ph AP 87.

2

Os AP 8

OsAP 8

Os A 8 Os A 8

OsAP 8

Os A 7 Os A 86

OsAP 8 Os A 8

7

OsA 9

OPhAsAP9 .2

PhA P9.1

0

PhAP

10.1

OsAP 44

OsAP 90

PhAP

44/9 1

PhA P44/

90.2

PhAP

44/9

0.3

41

PhAP

41.2

PhAP

41.1

OsAP8 8

PhAP

88.2

PhAP88 .4 PhAP88.

3 PhAP88.

5 PhAP88 .6 PhAP88 .7 PhAP88.8 OsAP6

71

95 98 100 99 100

100 100

76

71 100 10

0 98 99 100 99 99 100 74 10 0 100 1

0 100 10100 0 9 5

100 95 100 9 1 100 1 0

77

9

9 100 99

78 64

55

66

74

8

43

86 8

4 78 10 0 100

48

3

10 0

9 100 100

10 0

10

0 52100 95

100

99

100

100 94

62

10 0 100 10 0

69 7

100 10 0

100

49

100

5 99 99 1

98 100 100 100

76

100

100 100 100

100 100 100

100 100 100 100

84

99 99

69

100 100

70 95 100 100

99 100 100 100 100 100

83 100 93 95 100 100 100 100 100 100 100 100

71

85 69

100100 100 100

84

7 9 100 10 0 100

10076

68

99

99 100 100

90 93 77 97 96 96 100 100

100 75 5 100

8

100 97

83 85

100

84

10 0 78

100 1

00

100

100

83 85

83

99

100 100 98

1000

100

100 99 100100

96

100

75 69 92 96

98

96 97

93 95 93 95100 99

Fig 2 Phylogenetic tree of moso bamboo and rice aspartic proteinases Categories A, B and C are shaded pink, blue and purple, respectively The blue stars and green circles represent moso bamboo APs and rice APs, respectively The bootstrap percentages are shown close to the branch nodes

Chromosomal location and gene duplication events ofPhAPs

We mapped the PhAPs onto chromosomes to examine

the PhAP distribution on the moso bamboo

chromo-somes Among the 129 PhAP genes, 124 were located on

21 out of 24 moso bamboo chromosomes, while the

PhAPs was nonrandom but was scattered and uneven

Fourteen PhAPs located on chromosome 6 contained

the maximum number of PhAP genes; 13 PhAP genes were on chromosome 8; 12 PhAPs were on chromosome 14; and chromosomes 2, 5, and 11 had only one PhAP

duplications are considered to be the main reasons lead-ing to gene family expansion in plants As shown in

Chr1

0 25

Chr2

Chr3

0 25 50 75 100

Chr4

0 25 50

Chr5

0 25 50

Chr6

0 25 50 75 Chr7 0 25 50

Chr8

0 25 50 75

Chr9

0 25 50

Chr10 0 25 50

Chr1

25

25 50

50 75 100 125 Chr14

25 50 75 100

Chr15

0 25 50 75 100

Chr16

0 25 50

75

100

Chr17

0 25 50 75

100

Chr18

0

25

Chr19 0

25

Chr20 0

25

50

Chr21 0

25 50 75

10

0 25

50

Chr23 0

25

50 75

Chr24 0

25 50

PhAP44/90.2 PhAP44/90.1

P A P68 /76.1 PhAP44/90.3

PhAP58/73.1

PhAP8/21.3

PhAP8/21.2

PhAP8/21.4

PhAP8/21.1

PhA P45.2 PhAP19.2

PhA P22.2 PhAP39.1

PhAP71.1

PhAP16.2

PhAP12.1

PhAP 66.2

PhAP8

8.7

PhAP41.2

PhAP12.2 PhAP15.1

PhAP22.1

PhAP61.1 PhAP40.2

PhAP36.2

PhAP16.3

PhAP37.1

PhAP88.8

PhAP58.1 PhAP41.1

PhAP69.1

PhAP53.2 PhAP31.2

PhAP15.2

PhAP43.1

PhAP39.2

PhAP2 3.2 PhAP31.1

PhAP59.3

PhAP59.1 PhAP43.2

PhAP66.1

PhAP57.2 PhAP58.2

Ph AP65.2 PhAP52.1 PhAP74.2

PhAP10.1

PhAP19.1

PhAP23.1

PhAP57.5

PhAP45

.1

PhAP64.1

PhAP32.1

PhAP20.

1

PhAP50.1

PhAP74.1

Ph AP

71.2

PhAP2 5.1

PhAP65.1 PhAP27.1

PhAP53.1

PhAP16.1

PhAP32.2

PhAP25.2 PhAP88.5

PhAP50.2PhAP52.2 PhAP24.1

PhA P 72.1

PhAP93.5 PhAP88.2

PhAP56.3

PhAP50.3

PhAP72.2

PhAP48.1 PhAP20.2

PhAP 70.1

PhAP27.2

PhAP91.

1

PhAP14.1

PhAP14.2

PhAP87.2

PhAP57.1 PhAP57.3 PhAP57.4

PhAP88.4 PhAP70.2

PhAP12.3 PhAP18.1

PhAP31.3

PhAP36.1

PhAP72.3

PhAP93.

1 PhAP93.2PhAP93.4

PhAP93.6PhAP35.1 PhAP88.6

PhAP13.1 PhAP17.1

PhAP59.2

PhAP56.1

PhA sp3.1 PhAsp3.2

PhAsp2

.1

PhAsp2.2

PhAsp1.1 PhAP9.1

PhAP7.1 PhAP4.2

PhAP4.1

PhAP7.3 PhAP5.1

PhAP7.4

PhAP7.2

Fig 3 Chromosomal location and tandem duplicated genes among 124 PhAP genes A total of 124 out of 129 PhAPs were mapped onto the chromosomes on the basis of their physical location Chromosome numbers (Chr1- Chr24) are at the bottom of each chromosome The gray lines indicate duplicated blocks, while the red lines indicate duplicated PhAP gene pairs The genes listed in red font are segmentally duplicated, while tandemly duplicated genes are shaded in green

PhAP22.2 PhAP53.1 PhAP91.1 PhAP70.2 PhAP68/76.1 PhAP8/21.1 PhAP8/21.3 PhAP48.1 PhAP57.1 PhAP57.2 PhAP57.3 PhAP57.5 PhAP20.2 PhAP58.1 PhAP56.1 PhAP73.1 PhAP58/73.1 PhAP66.2 PhAP17.1 PhAP25.1 PhAP14.1 PhAP7.2 PhAP7.3 PhAP45.2 PhAP87.1 PhAP4.2 PhAP39.2 PhAP16.1 PhAP16.3 PhAP71.1 PhAP87.2 PhAP93.2 PhAP93.4 PhAP93.6 PhAP64.1 PhAP50.1 PhAP27.1 PhAP28.1 PhAP72.1 PhAP72.3 PhAP36.1 PhAP61.1 PhAP5.1 PhAP43.2 PhAP12.3 PhAP12.1 PhAP40.2 PhAP65.1 PhAP15.2 PhAP19.2 PhAP35.1 PhAP74.2 PhAP31.2 PhAP59.3 PhAP59.1 PhAP7.4 PhAP31.3 PhAP23.1 PhAP52.1 PhAsp3.2 PhAsp1.1 PhAsp2.1 PhAP41.1 PhAP9.1 PhAP10.1 PhAP44/90.3 PhAP44/90.1 PhAP88.7 PhAP88.6 PhAP88.1 PhAP88.4

0 100 200 300 400 500 600 700 800 900

0 4000 8000 12000 5'

Motif 9 Motif 5 Motif 4 Motif 2 Motif 10 Motif 3 UTR CDS

Fig 4 (See legend on next page.)