DNA RESEARCH 6, 13-19 (1999)
A Novel Subtilisin-like Protease Gene from Arabidopsis thaliana
is Expressed at Sites of Lateral Root Emergence
Leon W. NEUTEBOOM,1 LUZ M. VETH-TELLO, Olton R. CLUDESDALE, Paul J. J. HOOYKAAS,
and Bert J. van der ZAAL*
Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands
(Received 10 December 1998) Abstract
Differential screening of a cDNA library for mRNA species that specifically accumulate during auxin-induced lateral root formation in Arabidopsis thaliana led to the isolation of the AIRS cDNA clone. The corresponding single copy gene consists of 10 exons which encode a protein that possesses all the character-istics of subtilisin-like proteases. The promoter of the AIRS gene was fused to the gusA (/3-glucuronidase) reporter gene and introduced into Arabidopsis. Expression was almost completely restricted to the outer layers of the parental root at sites of lateral root emergence and could be observed even before protrusion of the newly formed root tip. In the presence of external auxin, GUS activity was visible throughout the parts of the root that are competent for lateral root formation. By digesting structural proteins in the extracellular matrix of cells located above sites of lateral root formation, AIRS might weaken cell-to-cell connections and thus facilitate lateral root emergence.
Key words: Arabidopsis thaliana; auxin; lateral root; subtilisin-like protease
1. Introduction
Plants can extend their root system by the forma-tion of lateral roots, and Arabidopsis thaliana provides a relatively simple system to study this phenomenom histologically.1 Lateral roots are derived from pericycle cells deep within the parental root tissue. Cell division is initiated in these cells, eventuating in the creation of a lateral root primordium which, as it grows, forces its way through the outer cell layers before final emer-gence takes place. Studies of mutants altered in lateral root formation2'3 indicate that the initiation and matura-tion of lateral roots are controlled by the plant hormone auxin.
We have previously shown that addition of the auxin 1-naphthaleneacetic acid (1-NAA) to Arabidopsis root cul-tures leads to a ca. 17-fold increase in the number of lat-eral root primordia within 48 hr.4 A differential screening
Communicated by Michio Oisni
* To whom correspondence should be addressed. Tel. +31-71-5274760, Fax. +31-71-5274999, E-mail: Zaal@Rulbim. LeidenUniv.nl
f Present address: Department of Plant Molecular Physiology, University of Hawaii at Manoa, St. John 503, Honolulu, HI 96822, USA
} The nucleotide sequence data reported will appear in the EMBL, GenBank and DDJB Databases under the accession number AF098632
approach has been exploited to isolate cDNA clones of which the corresponding mRNAs accumulate during this process.4 One of these cDNA clones, designated AIRS, encodes a protein that possesses all the characteristics of serine proteases belonging to the family of subtilisins.4 The typical catalytic triad of the amino acids aspartic acid, histidine and serine, together with the substrate binding site, can be found in the deduced amino acid se-quence of the AIR3 cDNA. In mammals, subtilisins are involved in the cleavage of prohormones and proproteins at specific sites composed of basic amino acids or "diba-sic sites."5 cDNA clones encoding subtilisin-like proteases have been isolated recently from plants,6"11 but it is not clear yet which function is fulfilled by these proteins. All plant subtilisin-like proteases are believed to be active extracellularly.
14
2. Materials and Methods
Subtilisin-like Protease Gene [Vol. 6.
2.1. Screening of a genomic library and cloning of the
AIR3 gene
A genomic library of Arabidopsis thaliana ecotype Columbia (kindly provided by J. T. Mulligan and R. W. Davis via the EU-BRIDGE Arabidopsis DNA Stock
Center, Koln, Germany) was screened using [32P]cDNA
inserts of AIR34 (accession number AF055848, isolated
from ecotype C24). Plaques generating positive sig-nals were purified and phage DNA was isolated. The genomic DNA inserts were analyzed by restriction en-zyme mapping and suitable fragments hybridizing to the cDNA probes were cloned in pBluescript SK+(II) (Stratagene, La Jolla, CA). All procedures were
essen-tially done according to Sambrook et al.12 Sequence
reac-tions and sequence analysis were performed as described
in Neuteboom et al.4
2.2. Construction of a promoter-gusA fusion
A plasmid harboring a part of the AIRS gene was used
for a polymerase chain reaction (PCR) amplification of a 1158-bp promoter fragment introducing a Nco I site at the ATG initiation codon. PCR was performed us-ing the M13-20 primer and a primer with the composi-tion 5'-GTGTTAGCTCCATGGTTCTTCTTC-3'. The following cycles were used: cycles 1-4, 1 min 94°C, 1 min 34°C, 2 min 72°C; cycles 5-29, 1 min 94°C, 1 min 44°C, 2 min 72°C; cycle 30, 1 min 94°C, 1 min 44°C, 10 min 72°C. The PCR fragment was digested with Hindlll and Nco I and was cloned into the Nco I-HindUI sites of
GusXX.13 An additional 6.0-kb Sac I-Hindlll fragment
was cloned into the 5' end of the Hindlll site of the 1158-bp AIRS promoter-gws.4 construct resulting in a 7.1-kb
AIRS promoter-gus A fusion. The PCR fragment and
fu-sion with the gusA gene were checked by sequencing. The 6.0-kb Sac l-Hindlll fragment contains phage polylinker sequences at the Sac I terminus. The AIRS
promoter-gusA construct was cloned into the Xba l/Xho I sites of
the wide host range vector pMOGACAT.13
2.3. Transformation to Arabidopsis thaliana
The construct was mobilized to Agrobactenum
turne-faciens strain MOG10114 using a triparental mating
procedure.15 Transformation to Arabidopsis thaliana
eco-type Columbia was performed as described by Vergunst
et al.16
2.4- Histochemical staining of transgenic plants
T2 transgenic seeds were surface-sterilized as described
earlier.4 The seeds were sown in a straight line on agar
so-lidified 1/2 MS17 medium containing 50 mg/1 kanamycin.
Seeds were pushed into the agar and subsequently ver-nalized for 4 days at 4°C in the dark to promote syn-chronized germination. Petri dishes containing the
trans-genic seeds were sealed with parafilm and positioned at an angle of 80° to allow roots to grow downwards. Seedlings were grown for 5 &ays under a 16-hr
illumi-nation cycle (100 /imol photons m~2 s"1) at 21°C. For
induction experiments, seedlings were transferred to six-well macroplates (Greiner) containing 4.0 ml of liquid 1/2 MS medium per well. After the addition of 1-NAA (1 /xM or 0.1 fiM), the plates were shaken at 90 rpm with a 5-cm displacement for 16 hr at 21°C. Histochemi-cal staining for GUS activities using 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc) was performed as described
by Jefferson.18
3. Results and Discussion
3.1. Isolation and sequence analysis of the AIR3 gene
The AIRS cDNA insert was used as a probe to screen a genomic library. Two phages were isolated which both contained a large portion of the same AIRS gene. The gene contains ten exons which encode a protein of 772 amino acid residues (Fig. 1). The predicted protein and the 3' untranslated region show minor differences with the protein predicted from the original AIRS cDNA clone and its 3' untranslated region as a result of a dif-ference in ecotype (the cDNA clone was isolated from ecotype C24, while the gene was isolated from ecotype Columbia). AIRS was found to be a single copy gene by Southern blot analysis (data not shown), despite the presence of other Arabidopsis subtilisin-like protease se-quences in the databases. Cross-hybridization with the other genes does not take place as high sequence sim-ilarity is restricted to the characteristic regions of the corresponding proteins. Remarkably, the exon/intron ar-rangement of the AIRS gene is not correlated with the characteristic regions of the protein (Fig. 1A). Moreover, the nucleotides which encode the highly conserved re-gion around the D residue, which is part of the catalytic triad, are separated by an intron (Fig. IB). Between ex-ons IV and V and between exex-ons V and VI (Fig. 1A), the
AIRS gene possesses two unusually large introns (1431
and 1761 bp, respectively). The latter intron, which can be found between position 3893 and 5654 in Fig. IB, contains a region, between position 4725 and 5424, that shows resemblance with a putative non-autonomous transposon that has been identified previously in the se-quences with the accession numbers AC002354, Z97335. Similar transposon-like sequences seem to be spread over all chromosomes (data not shown). The remaining in-trons do not share any significant homology with se-quences in the database.
L. W. Neuteboom et al. 1") Aim 1.6 kb TATA-box A T O _i n in iv TAA vuvin K x I ; II i n : iv : V ; VT i VIII vra ! ; b : H N ! ; AIR3 i l l transcribed region • _ • signal peptide k£J pro-region [ I mi ture protein aspartic acid
II histidine :atalytic triad coding regions
N asparagine, substrate binding site
B
1 2 1 3 6 1 120 132 1 4 1 1 1 5 6 1 1 6 8 1 AAATTAATAGTTATATATTTA.IAAATAATATTAAACCAAAACAAAATCAGAAAAACTTAAGTTTAAACTTAAAACACATCAAAATATAAATAGCTTTGTAAAACCGTTAAAAAGGAAAGA A A A T C A G A A A A A C C A A T G T G A J ^ C C A A T A T G T A A T T T G G T A T T A G T T A G A T T T A T A T T T A T A T A G C A A T T C A A C T C T T A A A T A A C A T T T T A G T T A G T A A T G T T T T T T T G C A A T G T T T G A A AAAAAAAAAAATGTTTTGCAGTGTTTGAAAATCTAGAAGGTCCCAAGCAAAACAATTTTTATCAAATCGCTGAGAAAAATTGGAAACTCAGTCTAGCCGGTCCAAATCAGACAAAATCTC TCAAATCGGTTAATTTCTGATTTAAAATTTTATATTTTCTAATGCTTAAGAGACGTATACTGTAGTACTTGGGATTTTGGATTTAATTCATCATCTTTTCGACCTTCAAGCTTGTCGTTT ATTTTTGACAATTTTCAGTTTGAACTCTATGAAATTTTGATTTTCTTCTAACTCAAAATCAAATTAATTAATTACATTGCTAGTTTTTTTAGTTTTCAAAGTGAATGGAATATAATTAGT AATACATATGACTAAACATATCAAAACTAATTTAATAAGTTTATAAAACCTAAGTTCCAACTAGX'CGTTTTACGCGCTTTGATATCTATCAAACTTTGAGTTATCGCCTAGCAATATTTT G A A A A T G A C T T A C C C T A G A A C J ^ A C T A A T A T C T T T A C T C A T C T A A T T T T C C A A A T T T T C C T T G T A A C T T G A T G C T G T A A T C C T C T A T T T T A A C G T A T C T A T C G T T G T G A C A T T T C C C T A T T TTCTTATACTACTCAGAAGATGTATCAATTGAATACAATTTTCTGACGAAAAAATATTTTCTTTTATATCGGCTCTTTGAAGTTCTAATTTATATCGATTGACAAAAAARAAArfAAAAAA AAAATTTTATACATAGTTTTTCCTCTCTAAGTATATAGAAGACAATGATAAGATTAGGACCAACAAACAGGTCATAAATTATCAGTTTTTCTGCCAAATATAGCATCAATAGAAACTCCA TTATTGGGACGCAATGTGACCACTTGAGCATCTTATCATTTTTATTTCAAAACCACCTTATTACACTGAGCTTCCCTTTTTTCAGCAACAAATTAATGTCAAATCAATTATTAAAAATAC A A T A T A A A T T C T G G G A G A A T A J ^ C A A G T A T T T A T T A G T C A T T A C A C A T A A A A A A C C G T A T T T T G G T G T T T C G T A G A A A G T G A T A T C A T A C T A G T C T A T A A T G T G T T T C A C A A A A A A A A A A AAAAACTTATATCAAAATCAAAJ\TTTCATGTTCTTGAAAAGATTTCAATATATTTTTTTTTTTTGT1CAATCGATATTAATATAGACTCAAACGACCCTACCCCAAAAGATATTGTAATTTC
AATATTTTATTAACATAAAAGATCGCACACGGTACAACCAAAATAGTTTTCGAAACCAATAAAAAAGGAAATAAAAACCGATTCTTTTTTTTTGCAATCCCATTATAAAGGAGCAACGCA
put. transcription .start 1 5' coding end of cDNA J
C AAG C CAC AAG T AAAAAGT GAJ \AGTCCAACAAAGATACAAAGAAGAAGAGAGAAGAAGAAGAAGAATGAAGCTAACACATAACTTCTCGTTTCTTCTTCTTCTTCTTCTTGTTCACATGA
M K H N V H M
GCTCGAAACACATTCTTGCTTCTAAAGATGTACAT T CATT AC AT T AC C C ATTTAC AC TATAACAAT CTTTTTTTCATT T C ACAT T C T T TAT CAACCAT T TCTCTTTGTGTGATTT CAGTC
S S K H I L A I J K D S
• signal peptide splicing site
GTCTTCATATGTGGTGTACTTCGGAGCTCACTCTCATGTCGGAGAGATAACAGAAGATGCCATGGATCGAGTCAAAGAAACTCACTACGACTTTCTTGGTTCCTTCACTGGAAGGTTCTC H H V D M D R K 1 8 0 1 1 9 2 1 TTAAACAAAACAACTCATTAA^VAACTTATTTAATATGTGAAACCATTTTCACTGACTTAAGTCTTGATAATGTGAAACCAGCCGTGAGAGAGCCACAGACGCCATTTTCTACTCATACAC R E R A T D A I F Y 5 Y T 2 0 4 1 2161 A A A G C A C A T C A A C G G A T T C G C T G C T C A T C T A G A C C A C G A C C T T G C T T A T G A A A T C T C C A G T A A G T A T A C A A A C A T A A C C A T T T T A T C T T C T T C T C T C T T T G T G C T T C A C A A G A T T T G A C T H I N G F A A H L D H D L A Y E I S C T T T T C T C T T T T C T T G A T T A T T C C T C A A G A A C A C C C T G A A G T T G T G r C T G T C T T C C C A A A C A A A G C T T T A A A G C T T C A C A C G A C T C G A T C A T G G G A C T T T T T G G G T T T G G A A C A T A A C T C K H N K K R W D H N 2281 2401 2521 2641 2761 2881 3001 3121 3241 3361 34B1 3601 3721 3841 3961 4081 4201 4321 4441 4561 4681 4801 4 921 5041 5161 5281 5401 5521 5641
putative pro-region splicing site •
T T A C G T T C C T T C C T C T T C C A T T T G G A G A A A G G C T A G G T T C G G A G A A G A T A C C A T T A T T G C A A A T C T C G A T A C A G G T T C G T T T T C T C A T C T T T T T T T T T T T T T T T A T C T C T C T T T G T A A T T Y V P S S S I W R K A^ K F G E D T I I A^ N L [D] T T A T A T T T C T T T A T T T C T T G G T C A A C C T T T G C A A C T T T G A G A G C C T T G A G A T G G G T T T T T G G G A A G A T A C G T A C C T A C T T T T A A A A C T A T A T A T T T C C C A A A C T T G C A T T A C A A G A A A T A T A T A T A T T C G T T G T G T A G A T T A S A T T A G C T A G T A G T T A A G A T C T C T A G A T G T C A G C A A G A A T C A A A T C G G C C A T A T A T A T A T G C A A G C A T G T G C G C A T A T G C A T A T T C A T T A A T A T G C A T A T T C A C T T C T C T T A A C T A G A T A G A C T T A T G G C A T T T C T T A T C T T T A A T A T T C T A T A C G T T G A C T G C A T C G T A T A G T T T G A C T T G A A T C A T A A A C A C T A A A T C T T T A T A A G T T G T A T T A T T T A T A C C A A A T T C T G T A G T A A A T A C T T T T C T T T C A A A A G T A G T T A C G A A A A C G A A T T A A A T A A A C A T T T A T C G A G A T A A T A A A T A T A T C A A A A A G C A C C T T C C A T A T A A A T T A C A G A A T T T T T G T T T T T G G A A T A G A A A A G T T G G T G A A T A A T T C A T C G T A C A A T T A A G G T G T T T T T G G C A C C A T G C A T T T T T C T T T T A A C A T A A A T T T A A T G C A T T T A C T G C A G G T A A C T T C A C T A G T A G T AGATATGAGTAATGTGTATTCTATATTAATTTAGTA'TTCATGAGTTATGACTTATGGACTAATAACTTAAATTTTCAAAAAAATCGTAAAAGTATAAATACCAAGTGGATAGTCTAGTGG C T T A G T A T C C T T T A G T G A A G A T T G T G C T A T A T A C A G A A T G T A C T C T A G T T T G A A C C T T C T T T A T T A G G A A T G T A T T T A T C A C A G G C T G G T C C T G G C C G A A A A A G A A G A C T A C G G G T C T A A A A G T T C C A A A A T T T C C T C A A T \ A T C A A A G A A A C T C T T G C A A T G C T C T T T A T A C T T G A A G C T G T A A C A G A T C T T A A C C A T T C T T G T A G A T G A T G A T G A A C C A A C T T T T A A A C A T G A T A A T C T A A T T C G A G A T T T T G T A A T G T A T T T T C G T A A T T C T T C G G T T T C A G C T A G A T G A T G T C A A G T G A T T A A T A A A A A G A A A A A G T T T G T A T T G G G T T A A T A G T T A T T G G T G T T T T T A G T T C G A A T T T C T T T T T C T T T C T C A T A A A ^ T C T T G G G A C T T A G G A T G A T A A A G T T G G G A T A T A C T T G A G A A C T A G A A C T T T T C A T A C C C T T C C T T T G A A T A A T A T A A T A T T G A T T C A G A A A A G C A A T A G A A G T T G C C A C A A C T A A A A T a ^ T T G G T T C A T T T G G T T G T C T G A T T A T G A G A C A C C A T T A T G T A T T T T T A T T T T T A G A T T T T A G T A A C T T A A A C C C A C T T A C T A T C T T T G T A T T A A T A A G A A T A T G T T G T T G A T T A T A A T T T C A T A A A C T A A T A T T T T A A A A T T T G A T T G T G T A T G T G T A A A A A G G T G T G T G G C C A G A A T C A A A G A G C T T T A G A G A T G A A G G C T T G G G A C C A A T T C C T T C A G V M P E S K S F R D E G L G P I P S AGATGGAAGGGAATATGTCAA-^ACCAAAAAGATGCCACTTTCCATTGCAATAGGTTCGTTTTCTCTCTTTTACAATTTTTATTCTTTCAAATTTGGTGTTCAATCATTTTCACGTTAAGT R W K G I C Q N Q K D A T F H C N R
16 Subtilisin-like Protease Gene [Vol. 6, t c 5 7 61 CTCTCTCCACCGCCGCCGGAGATTTTGTTCCTfifiAGTCAGCATCTTCGGCCAAGGCAACGGCACGGCCAAAGGTGGCTCCCCTCGCGCCAGAGTGGCCGCGTACAAAGTCTGCTGGCCGC T L S T A A G D F V P G V S I F G Q G N G T A K G G S P R A R V A A Y K V C W P 5 3 8 1 CAGTCAAAGGCAACGAATGCTACGACGCAGACGTCCTCGCTGCTTTCGACGCTGCCATACACGACGGAGCTGACGTCATATCCGTCTCTCTAGGCGGCGAGCCTACCTCGTTTTTTAACG P V K G N E C Y D A D V L A A F D A A I H D G A D V I S V S L G G E P T S F F N c 6 0 0 1 ACAGTGTCGCTATTGGTTCCTTTCACGCCGCTAAGAAGCGGATCGTGGTTGTTTGCTCTGCCGGAAACTCTGGACCGGCAGATAGTACGGTTTCTAA"GTCGCGCCATGGCAGATTACCG D S V A I G S F H A A K K R I V 1? V C S A G [ 9 j S G i > A D S T V S N V A P W Q I T a 6 1 2 1 TTGGTGCTAGCACCATGGACCGTGAGTTTGCTAGCAATCTTGTTCTCGGTAATGGAAAACATTACAAGGTTAGATATTAAGTCCAAGAATATGGATCATATCTCTATCTCAAATCTTGAA V G A S T M D R E F A S N L V L G N G K H Y K 62 4 1 AATGAATICTTGAAATTGAGCTTGAATCTTTTTCATTCGTAGGGACAAAGCTTGTCATCCACGGCATTGCCACATGCTAAGTTCTATCCAATTATGGCATCTGTAAATGCAAAAGCTAAG G Q S L S S T A L P H A K F Y P I M A S V N A K A K ~~5 A S A L D A Q L C K L G 6481 ATCGCTTGACCCTATAAAGACAAAGGGAAAGATATTAGTGTGTCTCAGAGGGCAGAACGGGAGGGTGGAAAAGGGTCGGGCCGTGGCTTTAGGGGGT(iGCATAGGGATGGTTCTTGAGAA S L D P I K T K G K I L V C L R G Q N G R V E K G R A V A L G G G I G M V L E N t g eg 6601 CACTTATGTCACCGGGAATGATTTACTCGCTGACCCGCATGTCCTTCCAGCCACACAGCTCACTTCTAAGGATAGTTTTGCTGTGTCAAGATATATCAGCCAAACCAAGTAGGATTCTTG T Y V T G N D L L A D P H V L P A T Q L T S K D S F A V S R Y I S Q T K S M T 6 7 2 1 ATTCACACTTTACCTAAACTTGTTTCAAGTTCTGAAAGAAAACTTTATTTTTTGATGTAGGAAGCCGATTGCACACATTAr:TCC:TTCAAGGACAGAT7TGGGACTAAAACCAGCGCCTGT K P I A H I T P S R T D L G L K P A P V 68 41 AATGGCTTCATTTTCTTCAAAAGGTCCAAGCATAGTGGCTCCTCAGATTCTGAAGCCTGACATAACTGCTCCTGGTGTGAGTGTGATCGCTGCCTACACTGGAGCAGTCTCTCCAACAAA M A S F S S K G P S I V A P Q I L K P D I T A P G V S V I . A A Y T G A V S P T N 6 9 6 1 CGAACAGTTTGATCCTCGACGACTTCTGTTCAATnCTATTTCAGGAACCTCTATGTCTTGTCCTCATATCTCTGGCATTGCGGGTCTTCTCAAAACTC:GTTATCCTTCTTGGAGCCCCGC £ 0 F 5 P R R L L F N A I S G T 0 M S C P H I S G I A G L L K T R Y P S H E f A 7081 AGCTATCCGCTCTGCCATCATGACTACCGGTAAGAATGTATTTCGATTCTAGAGCTTACACATTGTrGACAAAATTGAAGTTTAATTCTATTTTACCACTTAAAACACATTAACATCIAC A I R S A I M T T 7201 TGGTCCTGGACTTATAACCAAATCTTGTTTCTTCCATGGCCATGTTCTGTTTTCTTAGTTTTAGGTTGGAAATATTGGAATGAGTTTTCTTGAATCTTACAGTTTTTTCACACTTTGTGT c 7321 TTCCCTTTCAGCAACAATAATGGATGACATTCCTGGACCTATCCAAAATGCAACCAACATGAAGGCAACACCTTTCAGTTTTGGGGCAGGACACGTCCAACCAAATTTAGCTGTGAATCC A T I M D D I P G P I Q N A T N M K A T P F S F G A G H V Q P N L A V N P T c 74 41 CGGTCTCGTATACGATTTAGGCATCAAGGATTACCTCAACTTCTTATGCTCACTTGGATACAATGCATCACAAATCTCTGTATTCTCGGGCAACAACTTTACTTGTTCAAGCCCTAAAAT G L V Y D L G I K D Y L N F L C S L G Y N A S Q I S V F S G N N F T C S S P K I 7 5 6 1 CAGTCTTGTTAACCTCAACTATCCTTCCATCACAGTTCCAAACTTAACATCAAGCAAAGTCACCGTTTCAAGGACTGTTAAAAACGTTGGACGACCT'ICGATGTATACCGTCAAGGTGAA S L V N L N Y P S I T V P N L T S S K V T V S R T V K N V G R P S M Y T V K V N t c a t t c 7 681 TAACCCACAGGGTGTTTATGTCGCGGTGAAGCCAACGAGCTTGAATTTCACCAAAGTTGGAGAGCAAAAGACGTTTAAAGTGATCCTTGTGAAAAGTAAGGGAAATGTGGCTAAGGGCTA N P Q G V Y V A V K P T S L N F T K V G E Q K T F K V I L V K S K G N V A K G Y H L L a c g t 7 8 0 1 TGTGTTTGGAGAATTGGTTTGGTCAGACAAGAAGCATCGTGTGAGAAGTCCCATTGTGGTGAAGCTCTAACTTTGAATTTGAGTGCCAATTATGATATTTGATCTATGAAGTTTAGTTGC V F G E L V W S D K K H R V R S P 1 V V K L s t o p M A t atat a
7 9 2 1 CTATATGTTTGAATTTAAGGCTTACAAAGAATGGTGTAATGTTGTGCTGAT CATATAAAATAA. TACCTAGACTAGCACCCGTGCTACAACACAGGTTTCTAGTTCTTATTACTGA 8 1 4 1 TATAACGTTACCGCTTGGTATAAAGGACAATGGAGACATTACAGTTGTGAAAAAGTGGTGGAACCAACTCTTTAAACCTAAGTTAATGGTCTTCCAACAGTAGACCATCTTGTCTAATGC 82 61 CTTTGTATTTTTAATAGATTTCATTCTAAAAAAGACAAAAAAAAAAGAAAAAAAAAGAATGGATCC
Figure 1. The A1R3 gene. A, Genomic organization of the AIR3 gene (above) and distribution of the exons over the protein (below). B, Nucleotide and deduced amino acid sequence of AIR3 and flanking regions. Coding regions are underlined. The CAAT box, TATA box, translation start and stop are shown in boldface. Potential N-linked glycosylation sites are shown in boldface in the amino acid sequence. The putative signal peptide splicing site and pro-region splicing site are indicated by triangles. The reactive D, H and S region as well as the substrate binding site are boxed. The putative transcription start and the 5' coding end of the cDNA clone are indicated by arrows. The polyadenylation site is shown in bold italics. Differences at the nucleotide level between the cDNA clone (ecotype C24, accession number AF055848) and the genomic clone (ecotype Columbia) are indicated in italics above the sequence. The cDNA clone lacks two bases at position 7922 indicated by two dots above the sequence. At position 7972 and 7988 the cDNA clone possesses extra bases. Differences at the amino acid level are indicated below the amino acid sequence.
protease from Arabidopsis8 seems to be encoded by such dieted, complete plant subtilisin-like proteases encoded an intronless gene (AF065639). A variety of Arabidop- by cDNA clones (Fig. 2), including the latest entries in
sis ESTs have been submitted, but due to their un- the database (the SBT gent^s from Lycopersicon
esculen-faithful and partial sequencing these cannot easily be turn). The structural similarity of plant subtilisin-like designated to sequenced genes. It could thus be that proteases, which has been acknowledged previously,4'9'10 several of the intronless genes are pseudogenes created is not invalidated: like other eukaryotic subtilisin-like by integration events of mRNA-derived sequences. Also proteases, all proteins possess a putative signal peptide, in tomato, it has been shown that many subtilisin-like a pro-region and four well-conserved domains (in terms genes are present. Subtilisin-like proteases are appar- of both in amino acid composition and relative position ently widely distributed in many different plant species within the protein) that form the active site of the ma-and their activity seems to be required in an array of pro- ture protein. Unique for the plant members is the large
cesses, such eis microsporogenesis (LIM919), nodule for- spacing (ca. 230 amino acids) between the substrate
bind-mation (Agl28) and defense against pathogen infection ing site and the active serine residue compared to other (PR-P69A9 and PR-P69B10). eukaryotic subtilisin-like proteases. Also, the pro-region splicing site is different. In other eukaryotic
subtilisin-3.2. Plant subtilisin-like proteases consequently share like proteases this cleavage is believed to be achieved by
the same basic structure autolysis at dibasic sites.20 For PR-P69A,21 cucumisin6
The deduced amino acid sequence of the AIRS gene has a n d L I M Q l 9 t h e m a t u r e Pr o t e i n w a s determined to start been analyzed before.4 Here, we have used the predicted w i t h t w 0^ r ~ ™*d™3. At comparable positions,
. , „ „ \ . , , ,, .., ,, ., all other plant subtihsm-hke proteins also possess two
18= Subtilisin-like Protease Gene [Vol. 6.
A B
/ i ! x
///
N'"
Figure 3. Histochemical localization of AIR3 promoter-GUS activity. A, staining around lateral roots. B and C, details of staining at sites where lateral roots protrude or will protrude, viewed with Nomarski optics. D, GUS expression in a single isle at a position where activity in the pericycle is not visible. E, GUS expression in roots after 16 hr of 1 ^tM 1-NAA treatment.
splicing of the pro-region. The putative pro-region splic-ing sites are not preceded by pairs of basic amino acids. This might indicate that, in contrast with other eukary-otic subtilisin-like proteases, the plant subtilisin-like pro-teases studied so far have a broad substrate specificity. This hypothesis is supported by in vitro activity
mea-surements of cucumisin.22
3.3. Study of transgenic Arabidopsis harboring an AIR3 promoter-gusA fusion
Despite the structural similarities, plant subtilisin-like proteases are expressed in response to entirely different developmental and environmental stimuli. We investi-gated the expression pattern of AIR3 to learn more about the specific function of the AIRS protein during lateral root formation. The sequence flanking the ATG initia-tion codon at the 5' end has been determined. A consen-sus CAAT box and TATA box can be found respectively 126 bp and 77 bp upstream from the ATG. The promoter area lacks clear homology to any known (promoter) se-quence. Elements which are known to be important in
the promoters of diverse auxin-inducible genes23 cannot
be found in the promoter sequence shown in Fig. IB. An
AIR3 promoter-<7«s.4 construct was introduced into Ara-bidopsis by Agrobacterium tumefaciens-mcd'mted
trans-formation. Seedlings derived from three independent transformants with a 3:1 segregation for the kanamycin resistance gene in the T2 and from six additional indepen-dent transformants with multiple copies of the T-DNA were tested for GUS activity. In the absence of exoge-nous auxin, staining was visible in the outer layers of the parental root around lateral roots and at sites where
lat-eral roots were about to emerge (Fig. 3A, 3B and 3C). Even at very early stages of lateral root development, when cell division activity in the pericycle was barely or not detectable, GUS activity could be observed in the outer layers of the root, suggesting that expression pre-cedes the actual formation of the lateral root primordium (Fig. 3D). Upon addition of auxin, GUS expression in-creased along the length of the root except for the parts that are not competent for lateral root formation; i.e., the root tips, the meristematic regions and the zones directly behind these regions (Fig. 3E).
3-4- A possible role for AIR3 subtilisin-like protease
gene
So far, biochemical evidence for protease activity of the
AIRS protein is lacking. By comparison with other plant
subtilisin-like proteases, one is able to speculate about the role of AIRS. The plant subtilisin-like proteases that have been identified all possess a putative signal peptide for translocation over the endoplasmic reticulum and a pro-region, probably necessary to keep the protein inac-tive, while none of the proteins contains possible trans-membrane regions or any other known targeting signals. This suggests that all known plant subtilisin-like pro-teases, including AIRS, are active outside the cell. For some of these proteins it has been shown that the ma-ture, active forms are present extracellularly. The pro-cessed LIM9 protein for instance, has been identified in the locules of anthers by immunocytochemistry experi-ments and could be responsible for the degradation of cell
wall matrices in order to release microspores.19 The
inter-No. 1] L. W. Neuteboom et al. 19
cellular spaces of viroid-infected leaves21 and was shown
to interact with a cell wall matrix protein, named LRP.24
In analogy with other plant subtilisin-like proteins, the most likely substrates for AIRS are structural proteins in the cell wall.
3.5. Conclusion
We have isolated and characterized the first complete plant subtilisin-like protease g.ene, the AIRS gene, and established its expression pattern by construction of a promoter-gwsyl fusion which was analyzed in Arabidopsis seedlings. The AIRS protein has the same structure as all plant subtilisin-like proteins identified so far. The pro-moter activity at sites of lateral root emergence suggests that AIRS digests structural proteins in the extracellular matrix in order to weaken the tissue and facilitate lateral root emergence.
Acknowledgments: The authors would like to thank
Johan Pinas for assistance in preparation of Fig. 3.
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