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Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana
Journal of Genetics and Genomics (Formerly Acta Genetica Sinica) February 2007, 34(2): 138-150
Research Article
Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana Fenghua Xu, Chengming Fan, Yueqiu He① Key Laboratory of Plant Pathology of the Ministry of Education, Yunnan Agricultural University, Kunming 650201, China
Abstract: Chitinases (EC3.2.1.14), found in a wide range of organisms, catalyze the hydrolysis of chitin and play a major role in defense mechanisms against fungal pathogens. The alignment and typical domains were analyzed using basic local alignment search tool (BLAST) and simple modular architecture research tool (SMART), respectively. On the basis of the annotations of rice (Oryza sativa L.) and Arabidopsis genomic sequences and using the bio-software SignalP3.0, TMHMM2.0, TargetP1.1, and big-Pi Predictor, 25 out of 37 and 16 out of 24 open reading frames (ORFs) with chitinase activity from rice and Arabidopsis, respectively, were predicted to have signal peptides (SPs), which have an average of 24.8 amino acids at the N-terminal region. Some of the chitinases were secreted extracellularly, whereas some were located in the vacuole. The phylogenic relationship was analyzed with 61 ORFs and 25 known chitinases and they were classified into 6 clusters using Clustal X and MEGA3.1. This classification is not completely consistent when compared with the traditional system that classifies the chitinases into 7 classes. The frequency of distribution of amino acid residues was distinct in different clusters. The contents of alanine, glycine, serine, and leucine were very high in each cluster, whereas the contents of methionine, histidine, tryptophan, and cysteine were lower than 20%. Each cluster had distinct amino acid characteristics. Alanine, valine, leucine, cysteine, serine, and lysine were rich in Clusters Ⅰ to Ⅵ, respectively. Keywords: Chitinase; rice; Arabidopsis; secreted characteristics; phylogenetics
Chitinases (EC3.2.1.14), which are present in
exists only in vacuolar chitinases. Some chitinases
various organisms, catalyze the hydrolytic cleavage of
have one or more chitin-binding domains (CBD) fol-
the β-1, 4-glycosidic bond in biopolymers N- acetyl-
lowing the N-terminal signal region. In addition, there
glucosamine and are largely found in chitin of ar-
is a variable linking region between CBD and the
[1]
thropods . However, their endogenous substrate has [2]
not been found in plants . Chitinases use two different hydrolytic mechanisms: Substrate-assisted cataly-
catalytic domain. Each domain has distinct functions[5]. The classification system of plant chitinases has
and acid catalysis .
been revised several times[5]. Chitinases can generally
Chitinases have some domains based on their
be divided into two categories, endochitinases and
amino acid sequences. Typical plant chitinases have
exochitinases, with respect to their hydrolytic sites.
an N-terminal signal region, a main structural domain
Plant chitinases, however, are divided into seven
(or a catalytic domain), and a C-terminal region that
classes, Ⅰ–Ⅶ, on the basis of their structure, sub-
[3]
sis
[4]
Received: 2006-02-17; Accepted: 2006-05-31 This work was supported by the 863 Program (No. 2002AA245041), the National Natural Science Foundation of China (No. 30260006), and the R&D Foundation of Yunnan Province (No. 2003GP06). ① Corresponding author. E-mail: [email protected] www.jgenetgenomics.org
Fenghua Xu et al.: Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana
strate specificity[6], mechanisms of catalysis, and sen-
139
both the plants using bio-software.
sitivity to inhibitors. Class Ⅰ chitinase is further divided into two subclasses, Class Ⅰa and Class Ⅰb.
1
Class Ⅰa chitinases are acidic and have a C-terminal
1. 1
region with approximately six amino acids located in vacuole; Class Ⅰb chitinases are basic and secreted in apoplast[5,7,8] and do not have a C-terminal region. Chitinases of Classes Ⅰa, Ⅰb, Ⅱ,Ⅳ, Ⅵ, and Ⅶ belong to Class PR (pathogenesis-related proteins)-3, Class Ⅲ belongs to PR-8, and Class Ⅴ to PR-11[9]. In healthy plants, some forms of chitinases, both vacuolar and apoplastic, are produced continuously[10,11]. Production of chitinases is regulated by a variety of stress factors, both biotic and abiotic, including infection, wound, drought, cold, ozone, heavy -
metals, excessive salinity, and UV light[1, 10, 12 14]. In addition, phytohormones, such as ethylene, jasmonic acid, salicylic acid, auxin, and cytokinin, induce chitinase expression[2]. It is well known that chitinases are usually involved in active or passive defense against pathogens[11,12,15]. However, chitinases are also known to regulate growth and development by generating or -
degrading signal molecules[16 18] and through programmed cell death (PCD)[19,20]. Interestingly, apoplastic chitinases show antifreeze activity in monocotyledonous plants[21] but not in dicotyledonous plants. Therefore, these enzymes have been studied by several researchers. Rice (Oryza sativa ssp. japonica) and Arabidopsis thaliana are two model plants representing monocotyledons and dicotyledons, respectively, whose genomes have been sequenced completely, but the structure and function of their genes and proteins are still not clear. It will be useful to first predict the structure and function of proteins using bio-software before confirming them through experiments. For example, the analysis of chitinases in rice and Arabidopsis has not been reported so far. In this article, an attempt was made to predict the structure, function, and classification of chitinases in www.jgenetgenomics.org
Materials and Methods Materials
The sequences were downloaded from the web sites ftp://ftp.tigr.org/pub/data/Eukaryotic_Projects/ o_sativa/ annotation_dbs/pseudomolecules/version_ 2.0/all_chrs/all.pep for rice and http://www.arabidopsis.org/ for Arabidopsis. On the basis of description of the rice variety, Nipponbare and Arabidopsis thaliana genomes, the number of chitinases in the two plants was found to be 37 and 24, respectively. The chitinases studied in this article are putative chitinases, chitinases-like, and proteins with chitinase activity. To exactly predict the cluster chitinases from rice and Arabidopsis, the sequences of some known chitinases from other plants were downloaded from the NCBI protein database as standards. They include: Class Ⅰ: AAA62421 (acidic) (Zea mays), AAB23692 (acidic) (Dioscorea japonica), AAC24807 (Solanum tuberosum), AAD04295 (extracellular) (Vitis vinifera); Class Ⅱ: AAB96340 (S. tuberosum), CAB99486 (Hordeum vulgare subsp. vulgare), AAX83262 (Triticum aestivum), AAC36359 (Capsicum annuum); Class Ⅲ: CAA77657 (basic) (Nicotiana tabacum), CAA77656 (acidic) (N. tabacum), CAA76203 (Lupinus albus), BAC65326 (V. vinifera), AAO47731 (acidic) (Rehmannia glutinosa), AAM08773 (Oryza sativa), AAN37391 (C. annuum); Class Ⅳ: CAA74930 (Arabidopsis thaliana), BAD77932 (Cryptomeria japonica), AAQ10093 (V. vinifera), ABA39179 (Linum usitatissimum), AAM95447 (V. vinifera), AAB01665 (Brassica napus ); Class Ⅴ: AAM18075 (Momordica charantia), CAA54373 (N. tabacum); Class Ⅵ: P11218 (Urtica dioica); and Class Ⅶ: AAP80801 (Gossypium hirsutum). 1. 2
Methods
1.2.1
-
Overall analysis of the chitinases[22 24]
Four bio-software were used for the analysis of
140
Journal of Genetics and Genomics
secreted chitinases: SignalP3.0 (http://www.cbs.dtu. dk/services/SignalP) for prediction of signal peptides in proteins, TMHMM2.0 (http://www.cbs.dtu. dk/ services/TMHMM/) for prediction of transmembrane helices in proteins, TargetP1.1 (http://www.cbs. dtu.dk/ services/TargetP) for identification of the subcellular location, and big-Pi Predictor (http:// mendel.imp. univie.ac.at/sat/gpi/gpi_server.html) for analysis of GPI-anchor site. The alignment search was carried out using BLAST (basic local alignment search) from the web site, http://www.ncbi.nlm.nih.gov/BLAST/. The program used was blastp (Protein-protein BLAST), and the database nr was selected. Typical domains were analyzed using the on-line software from the web site, http://smart.embl.de/smart/set_mode.cgi?GENOMIC =1. 1.2.2
Phylogenic tree of chitinases.
The multiple protein sequences alignments were
遗传学报
Vol.34 No.2 2007
showed that most of the top matched proteins were chitinases, chitinase precursors, and chitinase-like proteins. In addition, the top-ho- mologous species of each ORF are different. 2.1.1 Signal peptides (SPs) and GPI-anchor in the chitinases SPs are responsible for targeting proteins to the endoplasmic reticulum (ER) for subsequent transport through the secretory pathway [40, 41]. However, not all chitinases had SPs at their N-termini. In rice and Arabidopsis genomes, two thirds or 25 and 16 chitinases, respectively, had SPs at the N-termini (Table 2). ORFs with SPs are listed in Table 1. The genes for secreted chitinases were dispersed on different chromosomes. The SP sequence had an average of 24.8 aa. The GPI anchor is usually found at the C-termini of the protein. The results obtained using Big-Pi Predictor, however, indicated that all chitinases of rice or Arabidopsis do not have this structure.
carried out using the program Clustal X from the web
2.1.2
site, http://bips.u-strasbg.fr/fr/Documentation/Clustal
When 37 rice and 24 Arabidopsis chitinases were analyzed with TMHMM2.0, it was found that 15 rice chitinases had one transmembrane domain, which was located within the first 40 N-terminal amino acids, and one, 9638.m03593 had two transmembrane helices at 12–34 and 139–161. Only 4 Arabidopsis proteins had transmembrane helices within the N-terminal amino acid region. Of all the 61 proteins in both rice and Arabidopsis, only 9638.m03593 had a real transmembrane domain at 139–161 site, because TMHMM, however, may not distinguish signal peptide from transmembrane domains in the region.
X/, and the parameters were auto-generated. The molecular
evolutionary
genetic
tree
through
the
Neighbor-joining method was constructed using MEGA3.1 from the web site, http://www.megasof tware.net/index.html. The phylogenic tree was tested using Bootstrap (1,000 replicates; seed = 64,238). Pairwise deletion was selected for gaps/missing data.
2 2. 1
Results Characteristics of the chitinases
On the basis of genome annotation, chitinases genes were found on all 11 chromosomes except on chromosome 7 of rice, whereas in Arabidopsis, the chitinases genes were found on all of the five chromosomes (Table 1). The length of the open reading frames (ORFs) ranged from 200 aa to 400 aa except for 9631.m02567, which had 127 aa. The average ORF length is 301 aa. The BLAST alignment search
2.1.3
Transmembrane domains in the chitinases
Subcellular location of the chitinases
Target P1.1 analysis of 61 chitinases showed that these enzymes could be divided into five categories according to their subcellular locations, i.e., chloroplast, mitochondrion, secretory pathway, other locations, and “unknown”. Most of the enzymes exist in the secretory pathway. In rice, only three chitinases are located inside mitochondrion; one inside chlorowww.jgenetgenomics.org
Fenghua Xu et al.: Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana
Table 1
141
Overall analysis of ORFs of rice and Arabidopsis
ORF
L
Top-matched clone
9629.m01787
290
AAC95376
Chitinase
399
3.00E-130 Cynodon dactylon
-
9629.m04512
301
AAB47176
PRm 3
411
1.00E-113 Zea mays
25
9629.m06349
296
AAQ21404
Chitinase III
376
5.00E-103 Medicago truncatula
26
9630.m03783
271
NP_191010
ATEP3; chitinase
350
5.00E-103 Arabidopsis thaliana
-
9631.m00319
256
CAA90970
Chitinase
323
3.00E-87
Vitis vinifera
27
9631.m02567
127
No
No
No
No
No
No
9631.m02983
326
ABD47583
Chitinase
537
3.00E-151 Musa × paradisiaca
-
9632.m02919
479
CAA54374
Chitinase V
158
6.00E-37
Nicotiana tabacum
28
9632.m03968
229
AAT40051
Chitinase
325
1.00E-87
Zea diploperennis
29
9632.m03974
288
AAT40036
Chitinase
379
1.00E-103 Tripsacum dactyloides
9633.m00400
295
AAD54935
Chitinase precursor
335
2.00E-90
9633.m01399
297
CAC87260
Top-matched protein
Putative xylanase inhibitor protein Putative xylanase inhibitor protein Putative xylanase inhibitor protein EndochitinaseI-antifreeze protein precursor
S
298
E-value
2.00E-79
Top-homologous species
29 -
Petroselinum crispum Triticum durum Triticum durum Triticum durum
R
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
30
268
1.00E-70
248
2.00E-64
411
2.00E-113 Secale cereale
30
Chitinase; BoCHI1
428
1.00E-118 Bambusa oldhamii
-
AAF04454
Chitinase
330
4.00E-89
-
320
ABD47583
Chitinase
481
2.00E-134 Musa × paradisiaca
323
ABD47583
Chitinase
514
2.00E-144 Musa × paradisiaca
277
5.00E-73
Triticum durum
9633.m01410
293
CAC87260
9633.m01415
297
CAC87260
9633.m03044
340
AAG53609
9633.m03045
299
AAR18735
9633.m03046
340
9634.m04963 9634.m04964
Poa pratensis
30 30
-
9636.m04120
315
CAC87260
Putative xylanase inhibitor protein
9636.m04169
316
NP_172076
ELP; chitinase
431
3.00E-119 Arabidopsis thaliana
-
9637.m02807
326
NP_172076
ELP; chitinase
446
6.00E-124 Arabidopsis thaliana
-
9638.m02367
307
ABD32310
Glycoside hydrolase, family 18
311
3.00E-83
Medicago truncatula
-
9638.m02374
288
Q9SLP4
Chitinase 1 precursor
306
8.00E-82
Tulipa bakeri
9638.m03591
261
CAA55345
Chitinase
412
9638.m03593
296
CAA55345
Chitinase
349
9639.m04440
304
CAC87260
9639.m04442
290
CAC87260
9639.m04443
292
CAC87260
9639.m04445
289
CAC87260
9639.m04446
284
CAC87260
www.jgenetgenomics.org
Putative protein Putative protein Putative protein Putative protein Putative protein
xylanase inhibitor xylanase inhibitor xylanase inhibitor xylanase inhibitor xylanase inhibitor
279 321 282 299 299
Hordeum 9.00E-114 vulgare Hordeum 7.00E-95 vulgare Triticum 1.00E-73 durum Triticum 2.00E-86 durum Triticum 1.00E-74 durum Triticum 8.00E-80 durum Triticum 1.00E-79 durum
turgidum
subsp.
30
31,32
vulgare
subsp.
-
vulgare
subsp.
-
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
30 30 30 30 30
142
Journal of Genetics and Genomics
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(Table 1 continued) ORF
L
Top-matched clone
9639.m04447
280
CAC87260
9639.m04448
304
CAC87260
9639.m04449
300
CAC87260
9639.m04450
301
CAC87260
9639.m04451
302
CAC87260
9640.m01849
336
CAC87260
At1g02360.1
272
AAD54936
Chitinase precursor
416
5.00E-115 Petroselinum crispum
-
At1g05850.1
321
AAP80801
ChitinaseⅦ precursor
507
3.00E-142 Gossypium hirsutum
-
At1g56680.1
280
CAA43708
Chitinase
226
6.00E-58
Brassica napus
33
At2g43570.1
277
AAK62047
Chitinase 4-like protein
436
4.00E-121 Brassica napus
-
At2g43580.1
265
CAA43708
Chitinase
437
2.00E-121 Brassica napus
33
At2g43590.1
264
CAA43708
Chitinase
468
1.00E-130 Brassica napus
33
At2g43600.1
273
CAA43708
Chitinase
210
4.00E-53
Brassica napus
33
At2g43610.1
281
CAA43708
Chitinase
291
2.00E-77
Brassica napus
33
At2g43620.1
283
CAA43708
Chitinase
287
3.00E-76
Brassica napus
33
At3g12500.1
322
AAF69777
Chitinase Ⅰ
616
4.00E-175 Arabis fecunda
34
At3g16920.1
333
AAQ56599
Chitinase-like protein
541
1.00E-152 Gossypium hirsutum
35
At3g47540.1
214
CAA43708
Chitinase
291
9.00E-78
33
At3g54420.1
273
CAA40474
Chitinase
408
1.00E-112 Phaseolus vulgaris
36
At4g01700.1
280
CAA57773
ChitinaseⅡ
428
1.00E-118 Arachis hypogaea
37
At4g19720.1
363
CAA55128
Chitinase/lysozyme
298
2.00E-79
Nicotiana tabacum
38
At4g19730.1
332
CAA54374
ChitinaseⅤ
253
1.00E-65
Nicotiana tabacum
39
At4g19740.1
289
CAA55128
Chitinase/lysozyme
185
2.00E-45
Nicotiana tabacum
38
At4g19750.1
362
CAA55128
Chitinase/lysozyme
318
2.00E-85
Nicotiana tabacum
38
At4g19760.1
365
CAA54374
ChitinaseⅤ
322
1.00E-86
Nicotiana tabacum
39
At4g19770.1
261
CAA54374
ChitinaseⅤ
223
6.00E-57
Nicotiana tabacum
39
CAA55128
Chitinase/lysozyme
356
9.00E-97
Nicotiana tabacum
38
443
5.00E-123 Nicotiana tabacum
38
344
4.00E-93
Nicotiana tabacum
38
603
3.00E-171
Arabidopsis lyrata subsp. petraea
-
At4g19800.1
398
Top-matched protein Putative protein Putative protein Putative protein Putative protein Putative protein Putative protein
xylanase inhibitor xylanase inhibitor xylanase inhibitor xylanase inhibitor xylanase inhibitor xylanase inhibitor
At4g19810.1
379
CAA55128
Chitinase/lysozyme
At4g19820.1
366
CAA55128
Chitinase/lysozyme
At5g24090.1
302
BAC11879
Acidic endochitinase
S 305 394 287 280 276 134
E-value
Top-homologous species
Triticum durum Triticum 2.00E-108 durum Triticum 3.00E-76 durum Triticum 5.00E-74 durum Triticum 7.00E-73 durum Triticum 5.00E-30 durum 2.00E-81
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
Brassica napus
R 30 30 30 30 30 30
9629.m01787 to 9640.m01849 are ORFs on Chromosome 1 to Chromosome 12 except for Chromosome 7 of rice in turn. At1g ?????.1 to At5g ?????.1 are ORFs on Chr.1 to Chr.5 of Arabidopsis in turn. L: length; S: score; R: reference; -: unpublished and direct submission.
www.jgenetgenomics.org
Fenghua Xu et al.: Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana
Table 2
143
Sequences of signal peptides in the ORFs of rice and Arabidopsis genomes
ORF 9629.m01787 9629.m04512 9629.m06349* 9630.m03783 9631.m02983 9632.m02919 9632.m03974* 9633.m01399 9633.m01415 9633.m03044 9633.m03046 9634.m04963 9634.m04964 9636.m04169 9637.m02807 9638.m02367 9638.m03591 9639.m04440 9639.m04442 9639.m04443 9639.m04445 9639.m04446 9639.m04447* 9639.m04449 9639.m04451 At1g05850.1 At1g56680.1 At2g43570.1 At2g43580.1 At2g43590.1 At2g43600.1 At2g43610.1 At2g43620.1 At3g12500.1 At3g16920.1 At3g47540.1 At3g54420.1 At4g01700.1 At4g19810.1 At4g19820.1 At5g24090.1
SPs length 31 26 20 25 21 24 29 21 24 32 32 18 20 27 28 25 29 26 27 28 26 21 21 31 29 26 28 31 24 24 22 28 28 20 27 20 28 21 24 22 22
C-domain and H-domain MAKPTPAPRATPFLLAAVLSIVVVAASG MAANKLKFSPLLALFLLAGIAVT MQMLIMVVVALAGLAAG MARRLSLLAVVLAMVAAVSAST MRALAVVAMVATAFLAAA MADKNGLLLLSTIAAVTLSSL MANSPTPTMLAFLALGLALLLSATGQ MASRRLAPLLVLLLSSSL MALRRHAALLSLAVVLLFAGL MSTPRAAASLAKKAALVALAVLAAALATA MIAARAANLQVAMKALALAVLALAYAAAT MRALALAVVAMAVVA MRALAVVVVATAFAVVA MRTSRAAAAAASLPLLLLVALLVA MKRKTRNKIILTTLLVSAAAILIGG MGSAKLIAVVLLPALLAFQAPM MTTTTTRFVQLAACAAASLLAVAASG MASQRRRSSATAVLLSLLLLLQL MGLVHALLPFAAAAALLLLAAPPP MAFGRRSLFLPVVGVAAILLLAAGH MAFRRRSCIPAALAVFFLLLAGQ MKMKALLPVAAMLLLVSG MMGLLSLLLVVVSCLAAP MASSSQRRRALPLSFVVIVLLILAGPGP MSKLQLRPPLLATLHCSLLVLLIING MVTIRSGSIVILVLLAVSFLALV MATQNKIQKNSLIIFLFTLVVIAQT MAKPTSRNDRFALFFITLIFLILTVSKP MALTKIFLILLLSLLGLYSET MAFTKISLVLLLCLLGFFSET MTIKNVIFSLFILAILAET MATQNAILKKALIIFLFTLTIMTGT MATLRAMLKNAFILFLFTLTIMAKT MKTNLFLFLIFSLLLSL MVSKPLFSLLLLTVALVVFQTGTL MASTKISLVFFLCLVGP MLTPTISKSISLVTILLVLQAFSNT MEKQISLLLCLLLFIFSI MSSTKLISLIVSITFFLTLQC MSSTKLISITFFLSLLLRF MTNMTLRKHVIYFLFFISC
N-domain AEA SRA ARA AAA VHA SLA ASA AAG AAA ARA ARA VRG VRG AEG TVA ATA AAA AAA ATA ATA STA QLA ATA VAG AAA ANG ATS VAS VKS VKS VFS AFS VFS SSA VNA CIG TKA SSS SMA SSA SLS
Cluster Ⅰ Ⅲ Ⅲ Ⅳ Ⅰ Ⅴ Ⅳ Ⅲ Ⅲ Ⅰ Ⅰ Ⅰ Ⅰ Ⅵ Ⅵ Ⅱ Ⅰ Ⅲ Ⅲ Ⅲ Ⅲ Ⅲ Ⅲ Ⅲ Ⅲ Ⅳ Ⅳ Ⅳ Ⅳ Ⅳ Ⅳ Ⅳ Ⅳ Ⅰ Ⅵ Ⅳ Ⅳ Ⅰ Ⅴ Ⅴ Ⅲ
The SPs are composed of C-domain, H-domain, and N-domain[42, 43]. The hydrophobic H-domain is indicated using gray shade. The SPs of ORFs with “*” do not have H-domain.
plast and seven at other locations. In Arabidopsis, only seven are at other locations and no chitinases are located inside mitochondrion and chloroplast.
listed in Table 3. These domains are Glyco_18, CBD, Pfam:Glyco_hydro_19, and Pfam:Glyco_hydro_18. All ORFs of Arabidopsis contain one or two kinds
2.1.4
of typical domains, whereas in rice, only 10 out of 36 ORFs have typical domains.
Typical domains in the chitinases
ORFs with the typical domain of chitinase are www.jgenetgenomics.org
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Journal of Genetics and Genomics
Table 3
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Vol.34 No.2 2007
ORFs with the typical domain of chitinase ORF
Typical domain
Begin
End
E-value
9629.m06349
Glyco_18
21
283
8.79E-01
9630.m03783
ChtBD1
27
58
1.75E-09
9631.m02983
ChtBD1
23
60
4.88E-17
9632.m02919
Glyco_18
41
389
1.27E-51
9632.m03974
ChtBD1
31
62
4.77E-11
9633.m03044
ChtBD1
34
71
3.90E-16
9633.m03045
ChtBD1
9
46
1.79E-15
9633.m03046
ChtBD1
34
71
3.96E-17
9634.m04963
ChtBD1
20
57
2.96E-16
9634.m04964
ChtBD1
22
59
1.46E-15
At1g02360.1
Pfam:Glyco_hydro_19
33
265
7.00E-133
At1g05850.1
Pfam:Glyco_hydro_19
63
304
2.70E-56
At1g56680.1
Pfam:Glyco_hydro_19
87
280
9.90E-58
At2g43570.1
ChtBD1
33
64
6.90E-11
Pfam:Glyco_hydro_19
80
277
9.90E-89
ChtBD1
26
57
1.40E-11
Pfam:Glyco_hydro_19
72
265
5.60E-100
ChtBD1
26
57
1.20E-10
Pfam:Glyco_hydro_19
71
265
5.90E-114
ChtBD1
24
59
5.50E-11
Pfam:Glyco_hydro_19
83
273
9.40E-49
ChtBD1
30
64
3.60E-10
Pfam:Glyco_hydro_19
91
281
9.80E-85
ChtBD1
30
64
3.10E-11
Pfam:Glyco_hydro_19
93
283
3.40E-82
At3g12500.1
ChtBD1
22
60
2.91E-15
At3g16920.1
Pfam:Glyco_hydro_19
71
313
1.30E-60
At3g47540.1
Pfam:Glyco_hydro_19
33
214
1.90E-69
At3g54420.1
ChtBD1
30
61
2.40E-09
At2g43580.1 At2g43590.1 At2g43600.1 At2g43610.1 At2g43620.1
Pfam:Glyco_hydro_19
75
273
2.50E-116
At4g01700.1
Pfam:Glyco_hydro_19
41
273
3.70E-135
At4g19720.1
Glyco_18
5
337
4.67E-100
At4g19730.1
Glyco_18
13
332
9.80E-77
At4g19740.1
Glyco_18
14
289
8.35E-47
At4g19750.1
Glyco_18
13
342
8.53E-94
At4g19760.1
Glyco_18
13
352
5.00E-95
At4g19770.1
Glyco_18
1
244
4.80E-47
At4g19800.1
Glyco_18
6
331
2.10E-106
At4g19810.1
Glyco_18
27
353
1.40E-111
At4g19820.1
Glyco_18
25
351
3.50E-99
At5g24090.1
Pfam:Glyco_hydro_18
30
289
1.90E-54
Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. ChtBD1: Chitin binding domain (CBD). www.jgenetgenomics.org
Fenghua Xu et al.: Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana
2. 2 2.2.1
145
Phylogeny of the chitinases Phylogenic tree of the chitinases
The phylogenic tree of the chitinases from rice, Arabidopsis, and partial genomic sequence of other plants as shown in Fig. 1 indicated that the chitinases originated from the same ancestor in the evolutionary history and then segregated into two branches; the one including the traditional Classes Ⅲ and Ⅴ and the other comprising the traditional Classes Ⅰ, Ⅱ, Ⅳ, Ⅵ, and Ⅶ. Surprisingly, the two branches share common features with the traditional classification. Chitinases from Classes Ⅲ and Ⅴ comprise the family 18 glycosyl hydrolases, and Classes Ⅰ, Ⅱ, Ⅳ, Ⅵ, and Ⅶ correspond to the family 19 enzymes[2,44–48].
However, the chitinases in the tree are
classified into 6 clusters, Clusters Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ, and Ⅵ (Fig. 1), which is not consistent with the traditional classification that has seven classes. Cluster Ⅰ is mainly composed of the traditional Classes Ⅰ , Ⅱ , and Ⅵ . Clusters Ⅲ , Ⅳ , and Ⅴ are equivalent to the traditional Classes Ⅲ, Ⅳ, and Ⅴ, respectively, whereas chitinase AAB23692 belonging to the traditional Class Ⅰ is grouped into Cluster Ⅳ. Cluster Ⅵ corresponds to traditional Class Ⅶ , whereas Cluster Ⅱ is a new group, which includes three
rice
chitinases,
among
which
chitinase
AMM08773 belongs to the traditional Class Ⅲ. The ORFs 9631.m02567 and 9640.m01849 of rice showed lower homology compared with other chitinases. The results of ProtFun 2.2 (http://www. cbs.dtu.dk/services/ProtFun) analysis showed that they are not enzymes, which may be attributed to their incorrect genome annotations. In addition, 9638.m3593 of rice is also identified as a nonenzyme, which may be attributed to the possible errors resulting from the use of the bio-software. In fact, it is highly homologous to ClusterⅠ, and in our phylogenic tree, 9638.m3593 is still considered the Cluster Ⅰ chitinase. www.jgenetgenomics.org
Fig. 1 Phylogenic tree of the chitinases from rice and Arabidopsis and standard ones from other plants The ORFs 9631.m02567 and 6940.m01849 enclosed in a rectangle are non-enzymes.
On the basis of all the chitinases that were analyzed in this study, it was found that the main clusters
146
Journal of Genetics and Genomics
of the chitinases in monocotyledons and dicotyledons differed from each other. The monocotyledon chitinases mainly consist of Clusters Ⅰ, Ⅱ, Ⅲ, and Ⅳ, whereas the dicotyledon chitinases consist of Clusters Ⅰ, Ⅲ, Ⅳ, Ⅴ, and Ⅵ. Clusters Ⅲ and Ⅵ shared by monocotyledons and dicotyledons are divided into two subclusters each, indicating that the chitinases in the same cluster are still evolving in different plants. Cluster Ⅰ, shared by monocotyledons and dicotyledons, is different from Clusters Ⅲ and Ⅵ because it has not been classified into two sub-clusters, although it has some differentiation. To better analyze the chitinases from rice and Arabidopsis, except for 9631.m02567 and 6940. m01849, they were extracted from the phylogenic tree (Fig. 1) and individually analyzed. Cluster Ⅲ mainly consists of the rice chitinases and only one Arabidopsis chitinase (At5g24090.1); Cluster Ⅳ mainly includes the Arabidopsis chitinases, with an exception
遗传学报
Vol.34 No.2 2007
2.2.2 Frequency of amino acid residues in the chitinases The frequencies of amino acid residues were calculated for all chitinases (Table 4). The distribution of amino acid residues differed with clusters. The frequencies of alanine, glycine, serine, and leucine in each cluster are 62.5%, 58.9%, 44.3%, and 42.4%, respectively; the frequencies of methionine, histidine, tryptophan, and cysteine are 10.0%, 10.5%, 12.3%, and 15.8%, respectively. Overall, cysteine, glycine, and proline mainly exist in Clusters Ⅰ and Ⅳ. It is well known that the CBD is rich in cysteine and the hinge region is rich in glycine and proline; as a result, Clusters Ⅰ and Ⅳ can contain the CBD and hinge region. Among the six clusters, each cluster is rich in one unique amino acid residue. Clusters Ⅰ to Ⅵ are rich in alanine, valine, leucine, cysteine, serine, and lysine, respectively.
3
Discussion
of 9632.m02919, which is rice chitinase. ClusterⅡ consists of 9638.m02367 and 9638.m02374 of rice. Cluster Ⅵ consists of two chitinases, each of rice and Arabidopsis. Thus, chitinases of rice and Arabidopsis are different. ClustersⅤ and Ⅳ are the main chitinases of Arabidopsis, whereas ClustersⅠ, Ⅱ, and Ⅲ are the main chitinases of rice. Therefore, it could be inferred that some chitinases could be specific for rice and Arabidopsis. Table 4 Cluster
3. 1
Characteristics of the secreted chitinases
The soluble extracellular-secreted proteins should have the following characteristics: (a) presence of an N-terminal signal peptide; (b) absence of transmembrane domains; (c) absence of GPI-anchor site; and (d) absence of localization signal to target the protein to mitochondria or other intracellular organelles[49]. Therefore, all chitinases do not have secretory characteristics and do not function extracellu-
Frequency of amino acid residues in every cluster Ala Cys Asp Glu Phe Gly
His
Ile
Lys Leu Met Asn Pro
Gln Arg
Ser
Thr
Val
Trp Tyr
Ⅰ
12.4 4.1
5.1
3.0
4.7 12.1 1.4
3.3
2.6
5.6
1.4
4.6
6.4
4.1
5.1
6.6
6.0
5.0
2.1
4.7
Ⅱ
11.6 0.5
5.8
3.6
6.6
1.4
5.8
3.4
6.4
1.3
5.7
5.1
4.0
2.8
6.5
6.4
8.9
1.5
5.2
Ⅲ
9.5
1.7
6.5
2.4
4.1 10.9 2.2
4.3
4.1
9.6
1.7
4.3
4.1
3.4
5.3
7.1
4.6
6.8
2.2
5.3
Ⅳ
9.2
5.3
3.7
3.5
5.8 11.5 1.2
4.7
4.0
5.7
1.6
6.4
4.4
3.9
4.5
7.3
6.3
4.9
1.4
4.7
Ⅴ
10.7 1.0
6.4
3.6
5.0
7.2
1.6
4.6
4.6
7.5
1.3
4.1
4.7
2.5
3.5 10.3 6.3
7.0
2.6
5.6
Ⅵ
9.1
3.2
5.2
4.9
4.4
9.4
2.7
4.0
6.3
7.6
2.7
4.6
4.9
3.5
2.8
5.0
2.5
5.5
Total
7.8
6.5
5.2
62.5 15.8 32.7 21.0 30.6 58.9 10.5 26.7 25.0 42.4 10.0 29.7 29.6 21.4 24.0 44.3 34.8 37.6 12.3 31.0
The black bold and italic numbers indicate the frequency ≥5.0 www.jgenetgenomics.org
Fenghua Xu et al.: Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana
147
larly. This indicates that the chitinases have certain intracellular locations. Chitinases are not only secreted extracellularly but also play a role inside vacuole[2,7,8]. All vacuolar chitinases have a C-terminal region.
chitinases are 60%–65% identical, whereas there is more than 70% similarity within the classes. The se-
3. 2
homologous to the chitinases discussed above and do
Structure and the classification of the chitinases
After sequencing the complete genome of rice and Arabidopsis, the existence of many chitinases was predicted using methods such as alignment with known chitinase sequences and structural and functional analyses. Some of the proteins that were predicted in this study, however, might not be chitinases or even enzymes. It is clear that the traditional classification of chitinases has become less satisfactory in view of new discoveries. Features like acidic or basic characteristics or the presence of N-terminal CBD hevein domains or vacuolar targeting signals are not very useful for the classification of chitinases[50, 51]. According to the most popular classification system described earlier, ClassⅠ chitinases consist of a cysteine-rich N-terminal (mainly at eight sites) and a chitin-binding hevein domain that has several highly conserved aromatic amino acid residues, which is separated from the catalytic domain by a proline and glycine-rich hinge region. Disulfide bonds form between two cysteine residues to preserve the protein fold. However, conserved aromatic amino acid residues and those with electric charge interact with sub-
quence structures of Class Ⅳ are 35%–50% identical with those of ClassⅠ and Class Ⅱ, and more than 60% within the class [50]. Class Ⅲ chitinases are not not have a CBD. ClassⅤ chitinases, which was first isolated from tobacco, have no sequence similarity with the other plant chitinases. Instead, they are related to bacterial exochitinases[53]. Clas Ⅵ chitinases, the UDA (Urtica dioica agglutinin) precursors, are generally similar to ClassⅠ or Ⅱ members, but are different from ClassⅠ in that they have two typical CBDs in their N-terminal region. ClassⅦ chitinases, sharing about 30% identity with ClassⅠ or Ⅱ chitinases, do not contain CBD and a hinge region and their sequences are much shorter [54]. In this article,not all of the chitinases fit this classification. They are divided into six clusters on the basis of protein sequence similarity. Most of them still coincided with the former classification. This shows that the former classification system of plant chitinases may be rather confusing and its classification criteria are nonsystematic. Therefore, it should be revised and improved further. The frequencies of distribution of main amino acid residues in six clusters of chitinases are distinct with each cluster rich in one unique amino acid residue. Therefore, it is presumed that this feature could
strates. Class Ⅳ chitinases are similar to ClassⅠ
be considered as a criterion for classification of chiti-
chitinases, except that their molecular size is smaller
nases. As the main clusters of chitinases in rice and
than that of ClassⅠ chitinases because of one dele-
Arabidopsis are clearly different, we presume that the
tion in the CBD, several deletions of approximately 22 amino acids in the catalytic domain, and incomplete C-terminal region. Therefore, their mature proteins have only 241–255 amino acids, but the mature
clusters of chitinases might also be distinct in monocotyledons and dicotyledons.
proteins of ClassⅠ chitinases have approximately
All ORFs with a CBD are in Clusters Ⅰ and Ⅳ, but not all ORFs of the two clusters have the CBD. As mentioned above, ClusterⅠ contains the traditional
300 amino acids[52]. ClassⅡ chitinases are homolo-
ClassⅠ chitinases with a CBD, and Cluster Ⅳ is
gous to those of ClassesⅠ and Ⅳ, but do not have a
equivalent to the traditional Class Ⅳ chitinases having the CBD with a deletion. This shows that consid-
CBD. The primary structures of ClassesⅠ and Ⅱ www.jgenetgenomics.org
148
ering CBD as a criterion for the traditional classification is practical and reasonable. Currently, many bio-software are available online. Although the bio-software used in this article are highly precise, each have their own merits and demerits. Therefore, the results obtained by biosoftware analyses may not be accurate. Further studies are required to shed more light on these questions. Acknowledgment: The authors thank Prof. Talekar NS, Plant Protection College, Yunnan Agricultural University, for his revision of the manuscript.
Journal of Genetics and Genomics
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Vol.34 No.2 2007
developing seeds. Planta, 2000, 210(4): 543-550. 12 Hamel F, Bellemare G. Characterization of a class I chitinase gene and of wound-inducible, root and flower-specific chitinase expression in Brassica napus. Biochim Biophys Acta, 1995, 1263(3): 212-220. 13 Hanfrey C, Fife M, Buchanan-Wollaston V. Leaf senescence in Brassica napus: expression of genes encoding pathogenesis-related protein. Plant Mol Biol, 1996, 30(3): 597-609. 14 Bishop JG, Dean AM, Mitchell-Olds T. Rapid evolution in plant
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molecular
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of
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in
plant-pathogen coevolution. Proc Natl Acad Sci USA, 2000, 97(10): 5322-5327. 15 Krishnaveni S, Liang GH, Muthukrishnan S, Manickam A. Purification and partial characterization of chitinases from
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水稻和拟南芥中几丁质酶的分析 许凤华, 范成明, 何月秋 云南农业大学植物病理重点试验室, 昆明 650201 摘 要：几丁质酶(EC3.2.1.14)是一种降解几丁质的糖苷酶, 广泛存在于各种生物体中, 并在植物中对病原真菌起重要抗性作 用。首先通过 BLAST 在 GenBank 中对其同源性进行搜索, 用 SMART 分析其结构。基于水稻和拟南芥的基因组注释, 借助 4 个生物学软件(SignalP3.0, TMHMM2.0, TargetP1.1 and big-Pi Predictor), 分析了水稻所有 37 条和拟南芥所有 24 条几丁质酶 序列, 发现有些几丁质酶都分泌到细胞外, 有些定位于液泡中, 水稻中仅 25 条和拟南芥中仅 16 条几丁质酶序列有信号肽, 这些信号肽的平均长度为 24.8aa。利用 Clustal X 和 MEGA3.1 两个生物软件分析了 59 条几丁质酶序列和 25 条标准几丁质 酶的系统发育关系, 这些几丁质酶可分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ 和 Ⅵ等 6 大类。这种聚类结果与几丁质酶传统分为 7 类有 些差异。通过对 6 大类中各个氨基酸残基的分析, 发现丙氨酸、甘氨酸、丝氨酸和亮氨酸的使用频率在每类中都非常高, 而 蛋氨酸、组氨酸、色氨酸和半胱氨酸均低于 20%。各大类中彼此之间的某些氨基酸使用频率明显不同, Ⅰ-Ⅵ分别富含丙氨 酸、缬氨酸、亮氨酸、半胱氨酸、丝氨酸和赖氨酸。 关键词：几丁质酶；水稻；拟南芥；分泌特性；系统发育 作者简介：许凤华(1981-), 女, 山东临沂人, 硕士研究生, 研究方向：分子植物病理学。E-mail: [email protected]
www.jgenetgenomics.org
Research Article
Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana Fenghua Xu, Chengming Fan, Yueqiu He① Key Laboratory of Plant Pathology of the Ministry of Education, Yunnan Agricultural University, Kunming 650201, China
Abstract: Chitinases (EC3.2.1.14), found in a wide range of organisms, catalyze the hydrolysis of chitin and play a major role in defense mechanisms against fungal pathogens. The alignment and typical domains were analyzed using basic local alignment search tool (BLAST) and simple modular architecture research tool (SMART), respectively. On the basis of the annotations of rice (Oryza sativa L.) and Arabidopsis genomic sequences and using the bio-software SignalP3.0, TMHMM2.0, TargetP1.1, and big-Pi Predictor, 25 out of 37 and 16 out of 24 open reading frames (ORFs) with chitinase activity from rice and Arabidopsis, respectively, were predicted to have signal peptides (SPs), which have an average of 24.8 amino acids at the N-terminal region. Some of the chitinases were secreted extracellularly, whereas some were located in the vacuole. The phylogenic relationship was analyzed with 61 ORFs and 25 known chitinases and they were classified into 6 clusters using Clustal X and MEGA3.1. This classification is not completely consistent when compared with the traditional system that classifies the chitinases into 7 classes. The frequency of distribution of amino acid residues was distinct in different clusters. The contents of alanine, glycine, serine, and leucine were very high in each cluster, whereas the contents of methionine, histidine, tryptophan, and cysteine were lower than 20%. Each cluster had distinct amino acid characteristics. Alanine, valine, leucine, cysteine, serine, and lysine were rich in Clusters Ⅰ to Ⅵ, respectively. Keywords: Chitinase; rice; Arabidopsis; secreted characteristics; phylogenetics
Chitinases (EC3.2.1.14), which are present in
exists only in vacuolar chitinases. Some chitinases
various organisms, catalyze the hydrolytic cleavage of
have one or more chitin-binding domains (CBD) fol-
the β-1, 4-glycosidic bond in biopolymers N- acetyl-
lowing the N-terminal signal region. In addition, there
glucosamine and are largely found in chitin of ar-
is a variable linking region between CBD and the
[1]
thropods . However, their endogenous substrate has [2]
not been found in plants . Chitinases use two different hydrolytic mechanisms: Substrate-assisted cataly-
catalytic domain. Each domain has distinct functions[5]. The classification system of plant chitinases has
and acid catalysis .
been revised several times[5]. Chitinases can generally
Chitinases have some domains based on their
be divided into two categories, endochitinases and
amino acid sequences. Typical plant chitinases have
exochitinases, with respect to their hydrolytic sites.
an N-terminal signal region, a main structural domain
Plant chitinases, however, are divided into seven
(or a catalytic domain), and a C-terminal region that
classes, Ⅰ–Ⅶ, on the basis of their structure, sub-
[3]
sis
[4]
Received: 2006-02-17; Accepted: 2006-05-31 This work was supported by the 863 Program (No. 2002AA245041), the National Natural Science Foundation of China (No. 30260006), and the R&D Foundation of Yunnan Province (No. 2003GP06). ① Corresponding author. E-mail: [email protected] www.jgenetgenomics.org
Fenghua Xu et al.: Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana
strate specificity[6], mechanisms of catalysis, and sen-
139
both the plants using bio-software.
sitivity to inhibitors. Class Ⅰ chitinase is further divided into two subclasses, Class Ⅰa and Class Ⅰb.
1
Class Ⅰa chitinases are acidic and have a C-terminal
1. 1
region with approximately six amino acids located in vacuole; Class Ⅰb chitinases are basic and secreted in apoplast[5,7,8] and do not have a C-terminal region. Chitinases of Classes Ⅰa, Ⅰb, Ⅱ,Ⅳ, Ⅵ, and Ⅶ belong to Class PR (pathogenesis-related proteins)-3, Class Ⅲ belongs to PR-8, and Class Ⅴ to PR-11[9]. In healthy plants, some forms of chitinases, both vacuolar and apoplastic, are produced continuously[10,11]. Production of chitinases is regulated by a variety of stress factors, both biotic and abiotic, including infection, wound, drought, cold, ozone, heavy -
metals, excessive salinity, and UV light[1, 10, 12 14]. In addition, phytohormones, such as ethylene, jasmonic acid, salicylic acid, auxin, and cytokinin, induce chitinase expression[2]. It is well known that chitinases are usually involved in active or passive defense against pathogens[11,12,15]. However, chitinases are also known to regulate growth and development by generating or -
degrading signal molecules[16 18] and through programmed cell death (PCD)[19,20]. Interestingly, apoplastic chitinases show antifreeze activity in monocotyledonous plants[21] but not in dicotyledonous plants. Therefore, these enzymes have been studied by several researchers. Rice (Oryza sativa ssp. japonica) and Arabidopsis thaliana are two model plants representing monocotyledons and dicotyledons, respectively, whose genomes have been sequenced completely, but the structure and function of their genes and proteins are still not clear. It will be useful to first predict the structure and function of proteins using bio-software before confirming them through experiments. For example, the analysis of chitinases in rice and Arabidopsis has not been reported so far. In this article, an attempt was made to predict the structure, function, and classification of chitinases in www.jgenetgenomics.org
Materials and Methods Materials
The sequences were downloaded from the web sites ftp://ftp.tigr.org/pub/data/Eukaryotic_Projects/ o_sativa/ annotation_dbs/pseudomolecules/version_ 2.0/all_chrs/all.pep for rice and http://www.arabidopsis.org/ for Arabidopsis. On the basis of description of the rice variety, Nipponbare and Arabidopsis thaliana genomes, the number of chitinases in the two plants was found to be 37 and 24, respectively. The chitinases studied in this article are putative chitinases, chitinases-like, and proteins with chitinase activity. To exactly predict the cluster chitinases from rice and Arabidopsis, the sequences of some known chitinases from other plants were downloaded from the NCBI protein database as standards. They include: Class Ⅰ: AAA62421 (acidic) (Zea mays), AAB23692 (acidic) (Dioscorea japonica), AAC24807 (Solanum tuberosum), AAD04295 (extracellular) (Vitis vinifera); Class Ⅱ: AAB96340 (S. tuberosum), CAB99486 (Hordeum vulgare subsp. vulgare), AAX83262 (Triticum aestivum), AAC36359 (Capsicum annuum); Class Ⅲ: CAA77657 (basic) (Nicotiana tabacum), CAA77656 (acidic) (N. tabacum), CAA76203 (Lupinus albus), BAC65326 (V. vinifera), AAO47731 (acidic) (Rehmannia glutinosa), AAM08773 (Oryza sativa), AAN37391 (C. annuum); Class Ⅳ: CAA74930 (Arabidopsis thaliana), BAD77932 (Cryptomeria japonica), AAQ10093 (V. vinifera), ABA39179 (Linum usitatissimum), AAM95447 (V. vinifera), AAB01665 (Brassica napus ); Class Ⅴ: AAM18075 (Momordica charantia), CAA54373 (N. tabacum); Class Ⅵ: P11218 (Urtica dioica); and Class Ⅶ: AAP80801 (Gossypium hirsutum). 1. 2
Methods
1.2.1
-
Overall analysis of the chitinases[22 24]
Four bio-software were used for the analysis of
140
Journal of Genetics and Genomics
secreted chitinases: SignalP3.0 (http://www.cbs.dtu. dk/services/SignalP) for prediction of signal peptides in proteins, TMHMM2.0 (http://www.cbs.dtu. dk/ services/TMHMM/) for prediction of transmembrane helices in proteins, TargetP1.1 (http://www.cbs. dtu.dk/ services/TargetP) for identification of the subcellular location, and big-Pi Predictor (http:// mendel.imp. univie.ac.at/sat/gpi/gpi_server.html) for analysis of GPI-anchor site. The alignment search was carried out using BLAST (basic local alignment search) from the web site, http://www.ncbi.nlm.nih.gov/BLAST/. The program used was blastp (Protein-protein BLAST), and the database nr was selected. Typical domains were analyzed using the on-line software from the web site, http://smart.embl.de/smart/set_mode.cgi?GENOMIC =1. 1.2.2
Phylogenic tree of chitinases.
The multiple protein sequences alignments were
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Vol.34 No.2 2007
showed that most of the top matched proteins were chitinases, chitinase precursors, and chitinase-like proteins. In addition, the top-ho- mologous species of each ORF are different. 2.1.1 Signal peptides (SPs) and GPI-anchor in the chitinases SPs are responsible for targeting proteins to the endoplasmic reticulum (ER) for subsequent transport through the secretory pathway [40, 41]. However, not all chitinases had SPs at their N-termini. In rice and Arabidopsis genomes, two thirds or 25 and 16 chitinases, respectively, had SPs at the N-termini (Table 2). ORFs with SPs are listed in Table 1. The genes for secreted chitinases were dispersed on different chromosomes. The SP sequence had an average of 24.8 aa. The GPI anchor is usually found at the C-termini of the protein. The results obtained using Big-Pi Predictor, however, indicated that all chitinases of rice or Arabidopsis do not have this structure.
carried out using the program Clustal X from the web
2.1.2
site, http://bips.u-strasbg.fr/fr/Documentation/Clustal
When 37 rice and 24 Arabidopsis chitinases were analyzed with TMHMM2.0, it was found that 15 rice chitinases had one transmembrane domain, which was located within the first 40 N-terminal amino acids, and one, 9638.m03593 had two transmembrane helices at 12–34 and 139–161. Only 4 Arabidopsis proteins had transmembrane helices within the N-terminal amino acid region. Of all the 61 proteins in both rice and Arabidopsis, only 9638.m03593 had a real transmembrane domain at 139–161 site, because TMHMM, however, may not distinguish signal peptide from transmembrane domains in the region.
X/, and the parameters were auto-generated. The molecular
evolutionary
genetic
tree
through
the
Neighbor-joining method was constructed using MEGA3.1 from the web site, http://www.megasof tware.net/index.html. The phylogenic tree was tested using Bootstrap (1,000 replicates; seed = 64,238). Pairwise deletion was selected for gaps/missing data.
2 2. 1
Results Characteristics of the chitinases
On the basis of genome annotation, chitinases genes were found on all 11 chromosomes except on chromosome 7 of rice, whereas in Arabidopsis, the chitinases genes were found on all of the five chromosomes (Table 1). The length of the open reading frames (ORFs) ranged from 200 aa to 400 aa except for 9631.m02567, which had 127 aa. The average ORF length is 301 aa. The BLAST alignment search
2.1.3
Transmembrane domains in the chitinases
Subcellular location of the chitinases
Target P1.1 analysis of 61 chitinases showed that these enzymes could be divided into five categories according to their subcellular locations, i.e., chloroplast, mitochondrion, secretory pathway, other locations, and “unknown”. Most of the enzymes exist in the secretory pathway. In rice, only three chitinases are located inside mitochondrion; one inside chlorowww.jgenetgenomics.org
Fenghua Xu et al.: Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana
Table 1
141
Overall analysis of ORFs of rice and Arabidopsis
ORF
L
Top-matched clone
9629.m01787
290
AAC95376
Chitinase
399
3.00E-130 Cynodon dactylon
-
9629.m04512
301
AAB47176
PRm 3
411
1.00E-113 Zea mays
25
9629.m06349
296
AAQ21404
Chitinase III
376
5.00E-103 Medicago truncatula
26
9630.m03783
271
NP_191010
ATEP3; chitinase
350
5.00E-103 Arabidopsis thaliana
-
9631.m00319
256
CAA90970
Chitinase
323
3.00E-87
Vitis vinifera
27
9631.m02567
127
No
No
No
No
No
No
9631.m02983
326
ABD47583
Chitinase
537
3.00E-151 Musa × paradisiaca
-
9632.m02919
479
CAA54374
Chitinase V
158
6.00E-37
Nicotiana tabacum
28
9632.m03968
229
AAT40051
Chitinase
325
1.00E-87
Zea diploperennis
29
9632.m03974
288
AAT40036
Chitinase
379
1.00E-103 Tripsacum dactyloides
9633.m00400
295
AAD54935
Chitinase precursor
335
2.00E-90
9633.m01399
297
CAC87260
Top-matched protein
Putative xylanase inhibitor protein Putative xylanase inhibitor protein Putative xylanase inhibitor protein EndochitinaseI-antifreeze protein precursor
S
298
E-value
2.00E-79
Top-homologous species
29 -
Petroselinum crispum Triticum durum Triticum durum Triticum durum
R
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
30
268
1.00E-70
248
2.00E-64
411
2.00E-113 Secale cereale
30
Chitinase; BoCHI1
428
1.00E-118 Bambusa oldhamii
-
AAF04454
Chitinase
330
4.00E-89
-
320
ABD47583
Chitinase
481
2.00E-134 Musa × paradisiaca
323
ABD47583
Chitinase
514
2.00E-144 Musa × paradisiaca
277
5.00E-73
Triticum durum
9633.m01410
293
CAC87260
9633.m01415
297
CAC87260
9633.m03044
340
AAG53609
9633.m03045
299
AAR18735
9633.m03046
340
9634.m04963 9634.m04964
Poa pratensis
30 30
-
9636.m04120
315
CAC87260
Putative xylanase inhibitor protein
9636.m04169
316
NP_172076
ELP; chitinase
431
3.00E-119 Arabidopsis thaliana
-
9637.m02807
326
NP_172076
ELP; chitinase
446
6.00E-124 Arabidopsis thaliana
-
9638.m02367
307
ABD32310
Glycoside hydrolase, family 18
311
3.00E-83
Medicago truncatula
-
9638.m02374
288
Q9SLP4
Chitinase 1 precursor
306
8.00E-82
Tulipa bakeri
9638.m03591
261
CAA55345
Chitinase
412
9638.m03593
296
CAA55345
Chitinase
349
9639.m04440
304
CAC87260
9639.m04442
290
CAC87260
9639.m04443
292
CAC87260
9639.m04445
289
CAC87260
9639.m04446
284
CAC87260
www.jgenetgenomics.org
Putative protein Putative protein Putative protein Putative protein Putative protein
xylanase inhibitor xylanase inhibitor xylanase inhibitor xylanase inhibitor xylanase inhibitor
279 321 282 299 299
Hordeum 9.00E-114 vulgare Hordeum 7.00E-95 vulgare Triticum 1.00E-73 durum Triticum 2.00E-86 durum Triticum 1.00E-74 durum Triticum 8.00E-80 durum Triticum 1.00E-79 durum
turgidum
subsp.
30
31,32
vulgare
subsp.
-
vulgare
subsp.
-
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
30 30 30 30 30
142
Journal of Genetics and Genomics
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(Table 1 continued) ORF
L
Top-matched clone
9639.m04447
280
CAC87260
9639.m04448
304
CAC87260
9639.m04449
300
CAC87260
9639.m04450
301
CAC87260
9639.m04451
302
CAC87260
9640.m01849
336
CAC87260
At1g02360.1
272
AAD54936
Chitinase precursor
416
5.00E-115 Petroselinum crispum
-
At1g05850.1
321
AAP80801
ChitinaseⅦ precursor
507
3.00E-142 Gossypium hirsutum
-
At1g56680.1
280
CAA43708
Chitinase
226
6.00E-58
Brassica napus
33
At2g43570.1
277
AAK62047
Chitinase 4-like protein
436
4.00E-121 Brassica napus
-
At2g43580.1
265
CAA43708
Chitinase
437
2.00E-121 Brassica napus
33
At2g43590.1
264
CAA43708
Chitinase
468
1.00E-130 Brassica napus
33
At2g43600.1
273
CAA43708
Chitinase
210
4.00E-53
Brassica napus
33
At2g43610.1
281
CAA43708
Chitinase
291
2.00E-77
Brassica napus
33
At2g43620.1
283
CAA43708
Chitinase
287
3.00E-76
Brassica napus
33
At3g12500.1
322
AAF69777
Chitinase Ⅰ
616
4.00E-175 Arabis fecunda
34
At3g16920.1
333
AAQ56599
Chitinase-like protein
541
1.00E-152 Gossypium hirsutum
35
At3g47540.1
214
CAA43708
Chitinase
291
9.00E-78
33
At3g54420.1
273
CAA40474
Chitinase
408
1.00E-112 Phaseolus vulgaris
36
At4g01700.1
280
CAA57773
ChitinaseⅡ
428
1.00E-118 Arachis hypogaea
37
At4g19720.1
363
CAA55128
Chitinase/lysozyme
298
2.00E-79
Nicotiana tabacum
38
At4g19730.1
332
CAA54374
ChitinaseⅤ
253
1.00E-65
Nicotiana tabacum
39
At4g19740.1
289
CAA55128
Chitinase/lysozyme
185
2.00E-45
Nicotiana tabacum
38
At4g19750.1
362
CAA55128
Chitinase/lysozyme
318
2.00E-85
Nicotiana tabacum
38
At4g19760.1
365
CAA54374
ChitinaseⅤ
322
1.00E-86
Nicotiana tabacum
39
At4g19770.1
261
CAA54374
ChitinaseⅤ
223
6.00E-57
Nicotiana tabacum
39
CAA55128
Chitinase/lysozyme
356
9.00E-97
Nicotiana tabacum
38
443
5.00E-123 Nicotiana tabacum
38
344
4.00E-93
Nicotiana tabacum
38
603
3.00E-171
Arabidopsis lyrata subsp. petraea
-
At4g19800.1
398
Top-matched protein Putative protein Putative protein Putative protein Putative protein Putative protein Putative protein
xylanase inhibitor xylanase inhibitor xylanase inhibitor xylanase inhibitor xylanase inhibitor xylanase inhibitor
At4g19810.1
379
CAA55128
Chitinase/lysozyme
At4g19820.1
366
CAA55128
Chitinase/lysozyme
At5g24090.1
302
BAC11879
Acidic endochitinase
S 305 394 287 280 276 134
E-value
Top-homologous species
Triticum durum Triticum 2.00E-108 durum Triticum 3.00E-76 durum Triticum 5.00E-74 durum Triticum 7.00E-73 durum Triticum 5.00E-30 durum 2.00E-81
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
turgidum
subsp.
Brassica napus
R 30 30 30 30 30 30
9629.m01787 to 9640.m01849 are ORFs on Chromosome 1 to Chromosome 12 except for Chromosome 7 of rice in turn. At1g ?????.1 to At5g ?????.1 are ORFs on Chr.1 to Chr.5 of Arabidopsis in turn. L: length; S: score; R: reference; -: unpublished and direct submission.
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Fenghua Xu et al.: Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana
Table 2
143
Sequences of signal peptides in the ORFs of rice and Arabidopsis genomes
ORF 9629.m01787 9629.m04512 9629.m06349* 9630.m03783 9631.m02983 9632.m02919 9632.m03974* 9633.m01399 9633.m01415 9633.m03044 9633.m03046 9634.m04963 9634.m04964 9636.m04169 9637.m02807 9638.m02367 9638.m03591 9639.m04440 9639.m04442 9639.m04443 9639.m04445 9639.m04446 9639.m04447* 9639.m04449 9639.m04451 At1g05850.1 At1g56680.1 At2g43570.1 At2g43580.1 At2g43590.1 At2g43600.1 At2g43610.1 At2g43620.1 At3g12500.1 At3g16920.1 At3g47540.1 At3g54420.1 At4g01700.1 At4g19810.1 At4g19820.1 At5g24090.1
SPs length 31 26 20 25 21 24 29 21 24 32 32 18 20 27 28 25 29 26 27 28 26 21 21 31 29 26 28 31 24 24 22 28 28 20 27 20 28 21 24 22 22
C-domain and H-domain MAKPTPAPRATPFLLAAVLSIVVVAASG MAANKLKFSPLLALFLLAGIAVT MQMLIMVVVALAGLAAG MARRLSLLAVVLAMVAAVSAST MRALAVVAMVATAFLAAA MADKNGLLLLSTIAAVTLSSL MANSPTPTMLAFLALGLALLLSATGQ MASRRLAPLLVLLLSSSL MALRRHAALLSLAVVLLFAGL MSTPRAAASLAKKAALVALAVLAAALATA MIAARAANLQVAMKALALAVLALAYAAAT MRALALAVVAMAVVA MRALAVVVVATAFAVVA MRTSRAAAAAASLPLLLLVALLVA MKRKTRNKIILTTLLVSAAAILIGG MGSAKLIAVVLLPALLAFQAPM MTTTTTRFVQLAACAAASLLAVAASG MASQRRRSSATAVLLSLLLLLQL MGLVHALLPFAAAAALLLLAAPPP MAFGRRSLFLPVVGVAAILLLAAGH MAFRRRSCIPAALAVFFLLLAGQ MKMKALLPVAAMLLLVSG MMGLLSLLLVVVSCLAAP MASSSQRRRALPLSFVVIVLLILAGPGP MSKLQLRPPLLATLHCSLLVLLIING MVTIRSGSIVILVLLAVSFLALV MATQNKIQKNSLIIFLFTLVVIAQT MAKPTSRNDRFALFFITLIFLILTVSKP MALTKIFLILLLSLLGLYSET MAFTKISLVLLLCLLGFFSET MTIKNVIFSLFILAILAET MATQNAILKKALIIFLFTLTIMTGT MATLRAMLKNAFILFLFTLTIMAKT MKTNLFLFLIFSLLLSL MVSKPLFSLLLLTVALVVFQTGTL MASTKISLVFFLCLVGP MLTPTISKSISLVTILLVLQAFSNT MEKQISLLLCLLLFIFSI MSSTKLISLIVSITFFLTLQC MSSTKLISITFFLSLLLRF MTNMTLRKHVIYFLFFISC
N-domain AEA SRA ARA AAA VHA SLA ASA AAG AAA ARA ARA VRG VRG AEG TVA ATA AAA AAA ATA ATA STA QLA ATA VAG AAA ANG ATS VAS VKS VKS VFS AFS VFS SSA VNA CIG TKA SSS SMA SSA SLS
Cluster Ⅰ Ⅲ Ⅲ Ⅳ Ⅰ Ⅴ Ⅳ Ⅲ Ⅲ Ⅰ Ⅰ Ⅰ Ⅰ Ⅵ Ⅵ Ⅱ Ⅰ Ⅲ Ⅲ Ⅲ Ⅲ Ⅲ Ⅲ Ⅲ Ⅲ Ⅳ Ⅳ Ⅳ Ⅳ Ⅳ Ⅳ Ⅳ Ⅳ Ⅰ Ⅵ Ⅳ Ⅳ Ⅰ Ⅴ Ⅴ Ⅲ
The SPs are composed of C-domain, H-domain, and N-domain[42, 43]. The hydrophobic H-domain is indicated using gray shade. The SPs of ORFs with “*” do not have H-domain.
plast and seven at other locations. In Arabidopsis, only seven are at other locations and no chitinases are located inside mitochondrion and chloroplast.
listed in Table 3. These domains are Glyco_18, CBD, Pfam:Glyco_hydro_19, and Pfam:Glyco_hydro_18. All ORFs of Arabidopsis contain one or two kinds
2.1.4
of typical domains, whereas in rice, only 10 out of 36 ORFs have typical domains.
Typical domains in the chitinases
ORFs with the typical domain of chitinase are www.jgenetgenomics.org
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Journal of Genetics and Genomics
Table 3
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Vol.34 No.2 2007
ORFs with the typical domain of chitinase ORF
Typical domain
Begin
End
E-value
9629.m06349
Glyco_18
21
283
8.79E-01
9630.m03783
ChtBD1
27
58
1.75E-09
9631.m02983
ChtBD1
23
60
4.88E-17
9632.m02919
Glyco_18
41
389
1.27E-51
9632.m03974
ChtBD1
31
62
4.77E-11
9633.m03044
ChtBD1
34
71
3.90E-16
9633.m03045
ChtBD1
9
46
1.79E-15
9633.m03046
ChtBD1
34
71
3.96E-17
9634.m04963
ChtBD1
20
57
2.96E-16
9634.m04964
ChtBD1
22
59
1.46E-15
At1g02360.1
Pfam:Glyco_hydro_19
33
265
7.00E-133
At1g05850.1
Pfam:Glyco_hydro_19
63
304
2.70E-56
At1g56680.1
Pfam:Glyco_hydro_19
87
280
9.90E-58
At2g43570.1
ChtBD1
33
64
6.90E-11
Pfam:Glyco_hydro_19
80
277
9.90E-89
ChtBD1
26
57
1.40E-11
Pfam:Glyco_hydro_19
72
265
5.60E-100
ChtBD1
26
57
1.20E-10
Pfam:Glyco_hydro_19
71
265
5.90E-114
ChtBD1
24
59
5.50E-11
Pfam:Glyco_hydro_19
83
273
9.40E-49
ChtBD1
30
64
3.60E-10
Pfam:Glyco_hydro_19
91
281
9.80E-85
ChtBD1
30
64
3.10E-11
Pfam:Glyco_hydro_19
93
283
3.40E-82
At3g12500.1
ChtBD1
22
60
2.91E-15
At3g16920.1
Pfam:Glyco_hydro_19
71
313
1.30E-60
At3g47540.1
Pfam:Glyco_hydro_19
33
214
1.90E-69
At3g54420.1
ChtBD1
30
61
2.40E-09
At2g43580.1 At2g43590.1 At2g43600.1 At2g43610.1 At2g43620.1
Pfam:Glyco_hydro_19
75
273
2.50E-116
At4g01700.1
Pfam:Glyco_hydro_19
41
273
3.70E-135
At4g19720.1
Glyco_18
5
337
4.67E-100
At4g19730.1
Glyco_18
13
332
9.80E-77
At4g19740.1
Glyco_18
14
289
8.35E-47
At4g19750.1
Glyco_18
13
342
8.53E-94
At4g19760.1
Glyco_18
13
352
5.00E-95
At4g19770.1
Glyco_18
1
244
4.80E-47
At4g19800.1
Glyco_18
6
331
2.10E-106
At4g19810.1
Glyco_18
27
353
1.40E-111
At4g19820.1
Glyco_18
25
351
3.50E-99
At5g24090.1
Pfam:Glyco_hydro_18
30
289
1.90E-54
Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. ChtBD1: Chitin binding domain (CBD). www.jgenetgenomics.org
Fenghua Xu et al.: Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana
2. 2 2.2.1
145
Phylogeny of the chitinases Phylogenic tree of the chitinases
The phylogenic tree of the chitinases from rice, Arabidopsis, and partial genomic sequence of other plants as shown in Fig. 1 indicated that the chitinases originated from the same ancestor in the evolutionary history and then segregated into two branches; the one including the traditional Classes Ⅲ and Ⅴ and the other comprising the traditional Classes Ⅰ, Ⅱ, Ⅳ, Ⅵ, and Ⅶ. Surprisingly, the two branches share common features with the traditional classification. Chitinases from Classes Ⅲ and Ⅴ comprise the family 18 glycosyl hydrolases, and Classes Ⅰ, Ⅱ, Ⅳ, Ⅵ, and Ⅶ correspond to the family 19 enzymes[2,44–48].
However, the chitinases in the tree are
classified into 6 clusters, Clusters Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ, and Ⅵ (Fig. 1), which is not consistent with the traditional classification that has seven classes. Cluster Ⅰ is mainly composed of the traditional Classes Ⅰ , Ⅱ , and Ⅵ . Clusters Ⅲ , Ⅳ , and Ⅴ are equivalent to the traditional Classes Ⅲ, Ⅳ, and Ⅴ, respectively, whereas chitinase AAB23692 belonging to the traditional Class Ⅰ is grouped into Cluster Ⅳ. Cluster Ⅵ corresponds to traditional Class Ⅶ , whereas Cluster Ⅱ is a new group, which includes three
rice
chitinases,
among
which
chitinase
AMM08773 belongs to the traditional Class Ⅲ. The ORFs 9631.m02567 and 9640.m01849 of rice showed lower homology compared with other chitinases. The results of ProtFun 2.2 (http://www. cbs.dtu.dk/services/ProtFun) analysis showed that they are not enzymes, which may be attributed to their incorrect genome annotations. In addition, 9638.m3593 of rice is also identified as a nonenzyme, which may be attributed to the possible errors resulting from the use of the bio-software. In fact, it is highly homologous to ClusterⅠ, and in our phylogenic tree, 9638.m3593 is still considered the Cluster Ⅰ chitinase. www.jgenetgenomics.org
Fig. 1 Phylogenic tree of the chitinases from rice and Arabidopsis and standard ones from other plants The ORFs 9631.m02567 and 6940.m01849 enclosed in a rectangle are non-enzymes.
On the basis of all the chitinases that were analyzed in this study, it was found that the main clusters
146
Journal of Genetics and Genomics
of the chitinases in monocotyledons and dicotyledons differed from each other. The monocotyledon chitinases mainly consist of Clusters Ⅰ, Ⅱ, Ⅲ, and Ⅳ, whereas the dicotyledon chitinases consist of Clusters Ⅰ, Ⅲ, Ⅳ, Ⅴ, and Ⅵ. Clusters Ⅲ and Ⅵ shared by monocotyledons and dicotyledons are divided into two subclusters each, indicating that the chitinases in the same cluster are still evolving in different plants. Cluster Ⅰ, shared by monocotyledons and dicotyledons, is different from Clusters Ⅲ and Ⅵ because it has not been classified into two sub-clusters, although it has some differentiation. To better analyze the chitinases from rice and Arabidopsis, except for 9631.m02567 and 6940. m01849, they were extracted from the phylogenic tree (Fig. 1) and individually analyzed. Cluster Ⅲ mainly consists of the rice chitinases and only one Arabidopsis chitinase (At5g24090.1); Cluster Ⅳ mainly includes the Arabidopsis chitinases, with an exception
遗传学报
Vol.34 No.2 2007
2.2.2 Frequency of amino acid residues in the chitinases The frequencies of amino acid residues were calculated for all chitinases (Table 4). The distribution of amino acid residues differed with clusters. The frequencies of alanine, glycine, serine, and leucine in each cluster are 62.5%, 58.9%, 44.3%, and 42.4%, respectively; the frequencies of methionine, histidine, tryptophan, and cysteine are 10.0%, 10.5%, 12.3%, and 15.8%, respectively. Overall, cysteine, glycine, and proline mainly exist in Clusters Ⅰ and Ⅳ. It is well known that the CBD is rich in cysteine and the hinge region is rich in glycine and proline; as a result, Clusters Ⅰ and Ⅳ can contain the CBD and hinge region. Among the six clusters, each cluster is rich in one unique amino acid residue. Clusters Ⅰ to Ⅵ are rich in alanine, valine, leucine, cysteine, serine, and lysine, respectively.
3
Discussion
of 9632.m02919, which is rice chitinase. ClusterⅡ consists of 9638.m02367 and 9638.m02374 of rice. Cluster Ⅵ consists of two chitinases, each of rice and Arabidopsis. Thus, chitinases of rice and Arabidopsis are different. ClustersⅤ and Ⅳ are the main chitinases of Arabidopsis, whereas ClustersⅠ, Ⅱ, and Ⅲ are the main chitinases of rice. Therefore, it could be inferred that some chitinases could be specific for rice and Arabidopsis. Table 4 Cluster
3. 1
Characteristics of the secreted chitinases
The soluble extracellular-secreted proteins should have the following characteristics: (a) presence of an N-terminal signal peptide; (b) absence of transmembrane domains; (c) absence of GPI-anchor site; and (d) absence of localization signal to target the protein to mitochondria or other intracellular organelles[49]. Therefore, all chitinases do not have secretory characteristics and do not function extracellu-
Frequency of amino acid residues in every cluster Ala Cys Asp Glu Phe Gly
His
Ile
Lys Leu Met Asn Pro
Gln Arg
Ser
Thr
Val
Trp Tyr
Ⅰ
12.4 4.1
5.1
3.0
4.7 12.1 1.4
3.3
2.6
5.6
1.4
4.6
6.4
4.1
5.1
6.6
6.0
5.0
2.1
4.7
Ⅱ
11.6 0.5
5.8
3.6
6.6
1.4
5.8
3.4
6.4
1.3
5.7
5.1
4.0
2.8
6.5
6.4
8.9
1.5
5.2
Ⅲ
9.5
1.7
6.5
2.4
4.1 10.9 2.2
4.3
4.1
9.6
1.7
4.3
4.1
3.4
5.3
7.1
4.6
6.8
2.2
5.3
Ⅳ
9.2
5.3
3.7
3.5
5.8 11.5 1.2
4.7
4.0
5.7
1.6
6.4
4.4
3.9
4.5
7.3
6.3
4.9
1.4
4.7
Ⅴ
10.7 1.0
6.4
3.6
5.0
7.2
1.6
4.6
4.6
7.5
1.3
4.1
4.7
2.5
3.5 10.3 6.3
7.0
2.6
5.6
Ⅵ
9.1
3.2
5.2
4.9
4.4
9.4
2.7
4.0
6.3
7.6
2.7
4.6
4.9
3.5
2.8
5.0
2.5
5.5
Total
7.8
6.5
5.2
62.5 15.8 32.7 21.0 30.6 58.9 10.5 26.7 25.0 42.4 10.0 29.7 29.6 21.4 24.0 44.3 34.8 37.6 12.3 31.0
The black bold and italic numbers indicate the frequency ≥5.0 www.jgenetgenomics.org
Fenghua Xu et al.: Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana
147
larly. This indicates that the chitinases have certain intracellular locations. Chitinases are not only secreted extracellularly but also play a role inside vacuole[2,7,8]. All vacuolar chitinases have a C-terminal region.
chitinases are 60%–65% identical, whereas there is more than 70% similarity within the classes. The se-
3. 2
homologous to the chitinases discussed above and do
Structure and the classification of the chitinases
After sequencing the complete genome of rice and Arabidopsis, the existence of many chitinases was predicted using methods such as alignment with known chitinase sequences and structural and functional analyses. Some of the proteins that were predicted in this study, however, might not be chitinases or even enzymes. It is clear that the traditional classification of chitinases has become less satisfactory in view of new discoveries. Features like acidic or basic characteristics or the presence of N-terminal CBD hevein domains or vacuolar targeting signals are not very useful for the classification of chitinases[50, 51]. According to the most popular classification system described earlier, ClassⅠ chitinases consist of a cysteine-rich N-terminal (mainly at eight sites) and a chitin-binding hevein domain that has several highly conserved aromatic amino acid residues, which is separated from the catalytic domain by a proline and glycine-rich hinge region. Disulfide bonds form between two cysteine residues to preserve the protein fold. However, conserved aromatic amino acid residues and those with electric charge interact with sub-
quence structures of Class Ⅳ are 35%–50% identical with those of ClassⅠ and Class Ⅱ, and more than 60% within the class [50]. Class Ⅲ chitinases are not not have a CBD. ClassⅤ chitinases, which was first isolated from tobacco, have no sequence similarity with the other plant chitinases. Instead, they are related to bacterial exochitinases[53]. Clas Ⅵ chitinases, the UDA (Urtica dioica agglutinin) precursors, are generally similar to ClassⅠ or Ⅱ members, but are different from ClassⅠ in that they have two typical CBDs in their N-terminal region. ClassⅦ chitinases, sharing about 30% identity with ClassⅠ or Ⅱ chitinases, do not contain CBD and a hinge region and their sequences are much shorter [54]. In this article,not all of the chitinases fit this classification. They are divided into six clusters on the basis of protein sequence similarity. Most of them still coincided with the former classification. This shows that the former classification system of plant chitinases may be rather confusing and its classification criteria are nonsystematic. Therefore, it should be revised and improved further. The frequencies of distribution of main amino acid residues in six clusters of chitinases are distinct with each cluster rich in one unique amino acid residue. Therefore, it is presumed that this feature could
strates. Class Ⅳ chitinases are similar to ClassⅠ
be considered as a criterion for classification of chiti-
chitinases, except that their molecular size is smaller
nases. As the main clusters of chitinases in rice and
than that of ClassⅠ chitinases because of one dele-
Arabidopsis are clearly different, we presume that the
tion in the CBD, several deletions of approximately 22 amino acids in the catalytic domain, and incomplete C-terminal region. Therefore, their mature proteins have only 241–255 amino acids, but the mature
clusters of chitinases might also be distinct in monocotyledons and dicotyledons.
proteins of ClassⅠ chitinases have approximately
All ORFs with a CBD are in Clusters Ⅰ and Ⅳ, but not all ORFs of the two clusters have the CBD. As mentioned above, ClusterⅠ contains the traditional
300 amino acids[52]. ClassⅡ chitinases are homolo-
ClassⅠ chitinases with a CBD, and Cluster Ⅳ is
gous to those of ClassesⅠ and Ⅳ, but do not have a
equivalent to the traditional Class Ⅳ chitinases having the CBD with a deletion. This shows that consid-
CBD. The primary structures of ClassesⅠ and Ⅱ www.jgenetgenomics.org
148
ering CBD as a criterion for the traditional classification is practical and reasonable. Currently, many bio-software are available online. Although the bio-software used in this article are highly precise, each have their own merits and demerits. Therefore, the results obtained by biosoftware analyses may not be accurate. Further studies are required to shed more light on these questions. Acknowledgment: The authors thank Prof. Talekar NS, Plant Protection College, Yunnan Agricultural University, for his revision of the manuscript.
Journal of Genetics and Genomics
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Vol.34 No.2 2007
developing seeds. Planta, 2000, 210(4): 543-550. 12 Hamel F, Bellemare G. Characterization of a class I chitinase gene and of wound-inducible, root and flower-specific chitinase expression in Brassica napus. Biochim Biophys Acta, 1995, 1263(3): 212-220. 13 Hanfrey C, Fife M, Buchanan-Wollaston V. Leaf senescence in Brassica napus: expression of genes encoding pathogenesis-related protein. Plant Mol Biol, 1996, 30(3): 597-609. 14 Bishop JG, Dean AM, Mitchell-Olds T. Rapid evolution in plant
chitinases:
molecular
targets
of
selection
in
plant-pathogen coevolution. Proc Natl Acad Sci USA, 2000, 97(10): 5322-5327. 15 Krishnaveni S, Liang GH, Muthukrishnan S, Manickam A. Purification and partial characterization of chitinases from
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水稻和拟南芥中几丁质酶的分析 许凤华, 范成明, 何月秋 云南农业大学植物病理重点试验室, 昆明 650201 摘 要：几丁质酶(EC3.2.1.14)是一种降解几丁质的糖苷酶, 广泛存在于各种生物体中, 并在植物中对病原真菌起重要抗性作 用。首先通过 BLAST 在 GenBank 中对其同源性进行搜索, 用 SMART 分析其结构。基于水稻和拟南芥的基因组注释, 借助 4 个生物学软件(SignalP3.0, TMHMM2.0, TargetP1.1 and big-Pi Predictor), 分析了水稻所有 37 条和拟南芥所有 24 条几丁质酶 序列, 发现有些几丁质酶都分泌到细胞外, 有些定位于液泡中, 水稻中仅 25 条和拟南芥中仅 16 条几丁质酶序列有信号肽, 这些信号肽的平均长度为 24.8aa。利用 Clustal X 和 MEGA3.1 两个生物软件分析了 59 条几丁质酶序列和 25 条标准几丁质 酶的系统发育关系, 这些几丁质酶可分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ 和 Ⅵ等 6 大类。这种聚类结果与几丁质酶传统分为 7 类有 些差异。通过对 6 大类中各个氨基酸残基的分析, 发现丙氨酸、甘氨酸、丝氨酸和亮氨酸的使用频率在每类中都非常高, 而 蛋氨酸、组氨酸、色氨酸和半胱氨酸均低于 20%。各大类中彼此之间的某些氨基酸使用频率明显不同, Ⅰ-Ⅵ分别富含丙氨 酸、缬氨酸、亮氨酸、半胱氨酸、丝氨酸和赖氨酸。 关键词：几丁质酶；水稻；拟南芥；分泌特性；系统发育 作者简介：许凤华(1981-), 女, 山东临沂人, 硕士研究生, 研究方向：分子植物病理学。E-mail: [email protected]
www.jgenetgenomics.org