Biol. Chem., Vol. 381, pp.

667 – 678, August 2000 · Copyright © by Walter de Gruyter · Berlin · New York

Two Differentially Regulated Class II Chitinases

from Parsley

Yvonne Ponatha, Heike Vollbergb, categories (Kombrink and Somssich, 1995): (i) immediate,
Klaus Hahlbrock and Erich Kombrink* early defense responses of the directly invaded plant cells,
starting with signal recognition and transduction and fre-
Max-Planck-Institut für Züchtungsforschung,
quently leading to hypersensitive cell death (HR); (ii) local
Abteilung Biochemie, Carl-von-Linné-Weg 10,
gene activation in the close vicinity of infection sites, resul-
D-50829 Köln, Germany
ting in the de novo synthesis of numerous secondary
products, including phytoalexins, in the reinforcement of
* Corresponding author
structural barriers, such as the cell wall, or in indirect
inhibition of the pathogen; (iii) systemic activation of genes
encoding pathogenesis-related (PR) proteins, including
Two distinct cDNA clones, PcCHI1 and PcCHI2, with
chitinases and 1,3--glucanases, which are directly or
high sequence similarity to plant chitinases were iso-
indirectly inhibitory towards pathogens and have been
lated from parsley (Petroselinum crispum), expressed
associated with the phenomenon of systemic acquired
in Escherichia coli, and the encoded proteins function-
resistance (SAR).
ally identified as endochitinases. Different expression
In several pathosystems, the initial responses of
patterns of the corresponding mRNAs and proteins in
pathogen-invaded or elicitor-treated plant cells occur
infected and uninfected parsley plants indicated dis-
within a few minutes and are rapidly followed by local gene
tinct roles of the two isoforms in both pathogen de-
activation (Somssich and Hahlbrock, 1998). In parsley,
fense and plant development. Infection of parsley leaf
various parts of the non-host resistance response to the
buds with Phytophthora sojae resulted in the rapid,
soybean pathogen, Phytophthora sojae, have been char-
transient and highly localized accumulation of PcCHI1
acterized both at the cytological/histological level in
mRNA and protein around infection sites, whereas
infected leaves (Schmelzer et al., 1989; Reinold and Hahl-
PcCHI2 mRNA and protein were systemically induced
brock, 1996) and at the molecular level in suspension-cul-
at later infection stages. Similar differences in the tim-
tured cells treated with a structurally defined oligopeptide
ing of induction were observed in elicitor-treated, sus-
elicitor derived from P. sojae (Hahlbrock et al., 1995;
pension-cultured parsley cells. In uninfected plants,
Somssich and Hahlbrock, 1998). Nearly all of the individ-
PcCHI1 mRNA was particularly abundant in the trans-
ual responses occurring in infected leaves were closely
mitting tract of healthy flowers, suggesting a role in
mimicked by the elicitor/cell culture system, underscoring
the constitutive protection of susceptible transmitting
the suitability of the latter for detailed molecular studies
tissue of the style against pathogen ingress and/or
which would not have been possible with intact plant
in the fertilization process, possibly by affecting pol-
tissue. These responses include rapid and transient
len tube growth. Localization of PcCHI2 mRNA and
changes in inorganic ion fluxes across the plasma mem-
protein in the parenchymatic collenchyme of young
brane (Nürnberger et al., 1994), the accumulation of reac-
pedicels may indicate a function in the constitutive
tive oxygen species (ROS) referred to as ‘oxidative burst’
protection of this tissue. In addition to such distinct
(Jabs et al., 1997), changes in the phosphorylation status
roles of PcCHI1 and PcCHI2 in preformed and induced
of various proteins (Dietrich et al., 1990), and an extensive
pathogen defense, both chitinases may have endoge-
reprogramming of both primary and secondary metabo-
nous regulatory functions in plant development.
lism at the gene expression level (Somssich et al., 1989;
Key words: Local and systemic gene activation /
Batz et al., 1998).
Pathogen defense / Petroselinum crispum / Phytophthora
The majority of genes studied so far in this context re-
sojae / Phylogeny reconstruction.
sponded very rapidly, probably reflecting at least in part
the design of the screening strategy for particularly early
Introduction events in pathogen defense. Among the few late respond-
ing genes that have been identified in parsley are those
Pathogen attack of plants triggers a large variety of encoding an enzyme specific for furanocoumarin (i. e.,
defense mechanisms that can be divided into three major phytoalexin) biosynthesis, S-adenosyl-L-methione:berg-
aptol O-methyltransferase, and two cell wall-associated
a
Present address: Qiagen, Max-Volmer-Str. 4 – 8, D-40724 Hil-
proteins, one hydroxyproline-rich glycoprotein and one
den, Germany. anionic peroxidase (Batz et al., 1998). While their local
b
Present address: Solvay Arzneimittel, Hans-Böckler-Allee 20, activation at infection sites has been demonstrated
D-30173 Hannover, Germany. (Schmelzer et al., 1989; Kawalleck et al., 1995), it is still un-
668 Y. Ponath et al.
known whether they are also systemically activated and
associated with systemic acquired resistance.
Systemic gene activation is followed by the accumula-
tion of a large variety of proteins, collectively referred to as
pathogenesis-related (PR) proteins. PR proteins have
been identified in numerous plants and are considered
to be ubiquitous in the plant kingdom (Kombrink and
Somssich, 1997; van Loon, 1999). They are presently
grouped into 14 different families (van Loon and van
Strien, 1999), a classification that is continuously updat-
ed. The best known and most extensively studied exam-
ples are members of the PR protein families 2 and 3, which
have been functionally identified as 1,3--glucanases and
chitinases, respectively, whereas others comprise mem-
bers with unknown biological functions.
Chitinases catalyze the hydrolytic cleavage of chitin, a
-1,4-linked homopolymer of N-acetyl-D-glucosamine
(GlcNAc) that constitutes major parts of the cell walls of
some, but not all, fungal pathogens and of the exoskeleton
of arthropods. Several purified plant chitinases have been
shown to inhibit the growth of some fungal pathogens,
particularly in combination with 1,3--glucanases (Mauch
et al., 1988; Sela-Buurlage et al., 1993; Graham and
Sticklen, 1994; Kombrink and Somssich, 1997). Further-
more, constitutive co-expression of genes encoding chiti-
nases and other PR proteins (e. g., 1,3--glucanase) yield-
ed transgenic plants with enhanced disease resistance
(Zhu et al., 1994; Jach et al., 1995; Jongedijk et al., 1995;
Kombrink and Somssich, 1997). However, contrary data
also exist (Neuhaus et al., 1991a; Nielsen et al., 1993). At
any rate, one major role of chitinases seems to be a pro-
tective one, owing either directly to their antimicrobial ac-
tivity or indirectly to the release of elicitor-active signal
molecules from pathogen surface structures that trigger
the activation of defense responses.
However, apart from being induced in response to in-
fection, chitinases are also expressed in an organ- and cell
type-specific manner during development of uninfected
plants, suggesting that they might have additional, as yet
unknown functions. Recently, certain chitinases were
shown to be constitutively expressed only in certain or-
gans or cell types, such as flowers or epidermal cells
(Wemmer et al., 1994; Ancillo et al., 1999). Little is known
about their endogenous functions, and a plant-derived
substrate has not been identified. Even for a chitinase that
has been demonstrated to rescue the carrot cell mutant
ts11 defective in embryo development, the mode of action
or the endogenous substrate(s) are unknown (de Jong et
al., 1992, 1993; Kragh et al., 1996).
Structural differences led to the distinction of five major
classes of plant chitinases of which only classes I, II and IV
are sequence-related (Kombrink and Somssich, 1997).
Class I chitinases contain an N-terminal ER localization
signal, a cysteine-rich chitin-binding (‘hevein’) domain, a
glycine/proline-rich hinge region, the most highly con-
served catalytic domain, and frequently a C-terminal
extension. In class II chitinases the hevein domain and
C-terminal extension are lacking, whereas class IV chiti-
nases comprise all structural features of class I, but are
smaller in size due to several deletions. A more detailed
account of the classification and nomenclature of plant
chitinases is given elsewhere (Neuhaus, 1999). Here we
report on the cloning, functional expression and charac-
terization of two divergent class II chitinases from parsley.
Although they belong to the same class according to the
presently used definition, they are differentially expressed
under a variety of conditions and are therefore likely to
serve different functions in both pathogen defense and
plant development.
Results
Isolation and Analysis of Chitinase cDNAs
Screening of two cDNA libraries, representing the mRNA
from 3-h and 20-h elicitor-treated parsley cells, with the
basic class I chitinase cDNA, ChtB3, from potato (Beer-
hues and Kombrink, 1994), yielded several cDNA clones
which were sequenced and assigned to two different
groups, PcCHI1 and PcCHI2, with about 60% identity in
nucleotide sequence. The PcCHI1 group was further sub-
divided in two closely related subgroups, PcCHI1-1 and
PcCHI1-2, with 98% sequence identity to one another.
Within each subgroup, represented by 6 and 3 cDNAs,
respectively, all members were identical in sequence and
varied only in length and poly(A) attachment site. By con-
trast, among 7 cDNAs of the PcCHI2 group that where
analyzed in detail, no differences in nucleotide sequence
were observed, but they also varied in length and poly(A)
attachment site. One full-length representative each from
the more abundant PcCHI1 subgroup and from the
PcCHI2 group was used for the following experiments.
When compared with the amino acid sequences avail-
able in databases, the deduced parsley chitinases exhib-
ited particularly high similarity with the class II chitinase
Chi2;1 from peanut (78% for PcCHI1 and 57% for PcCHI2;
Kellmann et al., 1996) and the flower-specific, basic class
II chitinase SK2 from potato (59% for PcCHI1 and 76%
for PcCHI2; Wemmer et al., 1994), whereas the similarity
with various acidic class II and basic class I chitinases,
such as potato ChtA2 and ChtB3 (Beerhues and Kom-
brink, 1994; Büchter et al., 1997), was lower. Figure 1 com-
pares PcCHI1 and PcCHI2 with three selected chitinases
bearing putative ER localization signals as well as several
amino acids that are highly conserved in all chitinases and
have been shown to be important for their three-dimen-
sional structures or catalytic activity (Holm and Sander,
1994; Iseli-Gamboni et al., 1998). Both parsley isoforms
lack the hevein domain and the C-terminal extension for
vacuolar targeting that distinguish class I from class II
chitinases (Kombrink and Somssich, 1997). PcCHI1, but
not PcCHI2, contains three potential glycosylation sites.
The most abundant one of several previously isolated,
elicitor-induced parsley chitinases, chitinase A (Kirsch et
al., 1993), was digested with endoproteinase Glu-C (V8
protease from Staphylococcus aureus) and one selected
 Chitinases from Parsley 669

from the corresponding region in PcCHI1. Furthermore,

allowing for processing according to von Heijne (1983),
the deduced mature PcCHI2 protein has a calculated Mr of
26 801 and a pI of 6.3, which is in reasonably good agree-
ment with experimentally derived values of Mr ≈ 30 000
and pI ≈ 5.3 for the purified chitinase A (Kirsch et al., 1993).
Finally, in protein extracts from cultured cells (Figure 2B) or
various organs of parsley plants (not shown) the PcCHI2-
specific antiserum labeled a single protein band with the
same Mr as chitinase A. Together, these results suggest
that chitinase A is encoded by PcCHI2.
It has to remain open whether PcCHI1 has a similar,
close relationship to one of the other previously character-
ized parsley chitinases (B and C) which were less abun-
dant than chitinase A (Kirsch et al., 1993) and were there-
fore not available in amounts sufficient for sequence
determination. While the presence of three potential gly-
cosylation sites in the deduced PcCHI1 protein (Figure 1)
would be in agreement with the fact that three of the four
purified type C chitinases, C1, C3 and C4, were glycosy-
lated (Kirsch et al., 1993), other deduced properties (Mr
27 260; pI 8.1) differ considerably from those estimated
for the type C chitinases (Mr 35 100 – 38 200; pI 3.5 – 3.8).
However, such estimates are probably imprecise for gly-
coproteins and do not necessarily preclude a close rela-

Fig. 1 Sequence Comparison of Different Types of Plant

Chitinase.
The amino acid sequences predicted for PcCHI1-1 and PcCHI2
from parsley (Petroselinum crispum) were aligned with the pre-
dicted sequences of basic class II chitinase StChtSK2 from pota-
to (Solanum tuberosum), which is specifically expressed in pistils
(Wemmer et al., 1994), and the acidic class II and basic class I
chitinases, StChtA2 and StChtB3, which are induced in potato
leaves by pathogen infection (Beerhues and Kombrink, 1994;
Büchter et al., 1997). Gaps were introduced for optimal align-
ment. Amino acids common to any 4 of the 5 sequences are shad-
ed. The putative cleavage sites of the N-terminal ER localization
signals and of the C-terminal extension in StChtB3 are marked
with arrows. The chitin-binding or hevein domain located at the N-
terminus of mature StChtB3 is marked with a dotted line. The po-
sitions of potential glycosylation sites in PcCHI1-1 are indicated
by boxes, with the hypothetical glycosyl residues () bound to
asparagine residues N139, N176 and N217. The amino acid se-
quence of a peptide fragment isolated from parsley chitinase A
(Kirsch et al., 1993) is underlined. Functionally important, con-
served amino acid residues of various plant chitinases, as identi-
Fig. 2 Induction of PcCHI1 and PcCHI2 mRNA and Protein in
fied from crystal structures, by mutagenesis or labeling (Verburg
Elicitor-Treated Parsley Cells.
et al., 1992; Holm and Sander, 1994; Hart et al., 1995; Andersen et
(A) Cultured parsley cells were treated with Pep25 elicitor for the
al., 1997; Iseli-Gamboni et al., 1998), are marked as follows: Glu
times indicated and total extractable RNA (10 g per lane) was
essential for catalysis (); Tyr of the catalytic site (); Asn forming
separated on agarose gels, blotted onto nylon membranes and
a hydrogen bond to the substrate (); Thr, Gly, Gln important for
hybridized with radiolabeled probes for PcCHI1, PcCHI2, and
maintenance of active site geometry ().
PcPAL. (B) Protein extracts from elicitor-treated parsley cells
were subjected to SDS-polyacrylamide gel electrophoresis (20
g per lane), transferred to nitrocellulose, and probed with puri-
peptide fragment was sequenced for comparison with fied antisera specific for PcCHI1 and PcCHI2 at dilutions of 1:200
PcCHI1 and PcCHI2. This sequence, GPIQISYNYNYG- and 1:500, respectively. (C) The specificity of the antisera is
PAGK, is identical with the region from G139to K154 in Pc- demonstrated on a separate blot containing the purified, heterol-
CHI2 (underlined in Figure 1) and differs in two positions ogously expressed proteins (0.1 g per lane).
670 Y. Ponath et al.

tionship. In protein extracts from cultured cells (Figure 2B)
or different organs of parsley plants (not shown), the
PcCHI1-specific antiserum reacted with several proteins
varying in abundance and ranging in Mr from 32 000 –
35 000, and in that respect resemble chitinases B and C.
Genomic DNA blot analysis using the PcCHI1 and Pc-
CHI2 cDNAs as probes confirmed the lack of cross-hy-
bridization under the same stringency conditions as used
below for RNA-blot analysis and indicated the occurrence
of small gene families comprising about 2 – 4 members
each per haploid parsley genome (Vollberg, 1994).
revealed that both recombinant chitinases had an appar-
ent Mr of 30 000 (not shown). Following renaturation, the
purified proteins were tested for chitinase activity using
[3H]chitin radiolabeled in the acetyl moieties as substrate.
Both proteins catalyzed the formation of GlcNAc
oligomers of varying lengths, but not of free acetic acid,
indicating endochitinase activity. Lysozyme activity to-
wards a bacterial cell-wall preparation could not be
demonstrated, in contrast to results obtained previously
with the purified plant cell-derived chitinase A (Kirsch et
al., 1993).

Functional Analysis Elicitor-Induced mRNA and Protein Accumulation

The 5 portions of the PcCHI1 and PcCHI2 cDNAs, encod-
ing putative ER localization signals (Figure 1), were re-
placed with a sequence coding for an N-terminal 6xHis
tag, MRGSHHHHHHGS, and the resulting constructs
combined with an IPTG-inducible promoter. The recombi-
nant proteins were expressed in Escherichia coli and
found to be localized in exclusion bodies. The proteins
were extracted under denaturing conditions, purified to
apparent homogeneity by affinity chromatography on Ni-
NTA columns and analyzed by gel electrophoresis which
The time courses of PcCHI1 and PcCHI2 mRNA and pro-
tein accumulation in elicitor-stimulated parsley cell sus-
pension cultures were established using the respective
full-length cDNAs for RNA-blot hybridization (Figure 2A)
and antisera raised against the His-tag-containing pro-
teins for immunoblotting (Figure 2B). The absence of
immunological cross-reactivity was verified in a separate
experiment (Figure 2C). The parsley- and E. coli-derived
PcCHI1 proteins displayed considerable differences in the
apparent Mr of 34 000 and 30 000, respectively, which

Fig. 3 In Situ Localization of PcCHI1 mRNA and Protein in Sections of Parsley Primary Leaf Buds Infected with P. sojae.
Infection sites were identified by their autofluorescence under UV light (A, B, C) and are marked with arrow heads. Adjacent sections were
hybridized with antisense RNA generated from PcCHI1 (D, E, F) or with the complementary sense RNA as control (G, H, I). The PcCHI1 pro-
tein was localized in a second set of sections with purified antiserum raised against the heterologously expressed protein (J, K, L, M); the
preimmune serum served as control (N, O. P, Q). The pathogen, P. sojae, was localized at infection sites in parallel sections using a specif-
ic antiserum raised against heat-inactivated zoospores (Jahnen and Hahlbrock, 1988). Shown here is one intermediate time point
postinoculation (R). Bar = 50 m.
 Chitinases from Parsley 671

were ascribed to glycosylation of the native, plant-derived

form (see above). For the corresponding PcCHI2 proteins
such differences were not observed.
PcCHI1 mRNA and protein accumulated rapidly and
transiently, with peaks occurring around 2 and 6 – 8 h after
elicitor application, respectively, whereas increases in
PcCHI2 mRNA and protein commenced only after about
14 h and continued for prolonged periods of time (Figure
2A and B). Throughout this latter period, PcCHI1 mRNA
and protein remained present at moderate levels, similar
to PAL mRNA which was measured as a control and ex-
hibited a pronounced biphasic behavior (Batz et al., 1998).

mRNA and Protein Accumulation Around

Infection Sites
To test whether the elicitor-induced increases in cultured
cells mimicked similar effects at true infection sites, pars-
ley leaf buds were inoculated with P. sojae spores and
analyzed by in situ RNA/RNA hybridization and immuno-
histochemistry under previously established conditions
(Schmelzer et al., 1989; Reinold and Hahlbrock, 1996).
Figure 3 shows the results for PcCHI1 at various time in-
tervals postinoculation that had been selected on the ba-
sis of the accumulation patterns obtained with elicitor- Fig. 4 Immunolocalization of PcCHI2 Protein in Parsley Primary
Leaf Buds Infected with P. sojae.
treated cells. In contrast to treatment with elicitor, howev-
Sections were incubated with purified antiserum raised against
er, only approximate time spans can be given for the asyn-
PcCHI2 (A, B, C) or with preimmune serum as control (D, E, F).
chronously developing infections, and a few hours were Infection sites are marked with arrow heads and were identified
allowed for spore germination prior to the actual beginning in parallel sections by their autofluorescence under UV light. e,
of the infection process. Again, the induction was strong, epidermis; x, xylem. Bar = 50 m.
transient and consecutive for mRNA and protein. Both
were highly localized around infection sites which were
identified as UV-fluorescing (Figure 3B and C), brown
necrotic spots that greatly increased in size with time
postinoculation (Figure 3P and Q). For one selected inter-
mediate time point, the invading pathogen was shown by
immunolocalization to be sharply confined to a compara-
tively small area (Figure 3R). While PcCHI1 mRNA was
highly abundant at 10 – 12 h and had largely disappeared
at 20 – 25 h, the encoded protein was clearly present at
both time points and beyond.
Analogous in situ hybridization experiments failed to
demonstrate any appreciable accumulation of PcCHI2
mRNA at infection sites during the time period analyzed in
Fig. 5 Accumulation of PcCHI1 and PcCHI2 mRNA in Parsley
Figure 3A-I, possibly due to a slow, more or less uniform
Primary Leaves after Infection with P. sojae.
increase throughout the tissue, which would have been Total extractable RNA was isolated at the indicated times
difficult to verify unequivocally against a considerable postinoculation, separated on agarose gels (10 g per lane), blot-
background (data not shown). More clear-cut results were ted onto nylon membranes and hybridized with radiolabeled
obtained for the PcCHI2 protein at late time points (Fig- probes for PcCHI1 and PcCHI2.
ure 4). In contrast to PcCHI1, PcCHI2 was constitutively
expressed in the upper epidermis and in young xylem cells
Tissue-Specific Expression
(Figure 4A) and appeared to increase moderately in a
sharp zone around infection sites (Figure 4B and C) as well The large difference in PcCHI1 and PcCHI2 mRNA abun-
as systemically throughout the infected leaf (Figure 4C). dance in uninfected control leaves (0 h in Figure 5) prompt-
This latter indication of a late systemic induction would be ed us to further analyze their organ- and tissue-specific
in line with a slow, moderate overall increase of PcCHI2 expression. Figure 6 shows the results obtained for the
mRNA in infected parsley leaves, as compared with a major organs of 30-day-old seedlings. Again, while Pc-
much more rapid and transient accumulation of PcCHI1 CHI1 mRNA was moderately abundant in primary leaves,
mRNA in the same tissue (Figure 5). PcCHI2 mRNA was not detectable under these condi-
672 Y. Ponath et al.

tions. Based on equal loading of total RNA, PcCHI1 mRNA

Fig. 6 Relative PcCHI1 and PcCHI2 mRNA Levels in Different
Parsley Organs.
Seedlings were grown for 30 days in hydroculture. Total ex-
tractable RNA (10 g per lane) was separated on agarose gels,
blotted onto nylon membranes and hybridized with radiolabeled
probes for PcCHI1 and PcCHI2.
was by far most abundant in roots and to a lesser extent in
cotyledons, whereas PcCHI2 mRNA was most strongly
expressed in cotyledons and hypocotyls. A similar relative
mRNA distribution was observed in the corresponding
organs of 3-month-old plants (not shown).
Two organs of adult, 2-year-old plants, flower and stem
(pedicel), whose elaborate anatomical and functional dif-
ferentiation has previously yielded particularly well-struc-
tured distribution patterns for phenylpropanoid-related
mRNAs (Reinold and Hahlbrock, 1997), were analyzed
in greater detail by in situ hybridization. Signals strong
enough for unequivocal mRNA localization were obtained
for PcCHI1 in two different flower parts, style and trans-
mitting tract (Figure 7A and B), and for PcCHI2 in the
parenchymatic collenchyme of a young pedicel (Fig-
ure 7E).

Fig. 7 In Situ Localization of PcCHI1 mRNA in Flowers and PcCHI2 mRNA in Pedicels of Parsley.
Flowers were sectioned at two horizontal levels, either through the style (1; A, C) or through the ovary (2; B, D), as indicated in the schemat-
ic drawing. Flower sections were hybridized either with PcCHI1 antisense RNA (A, B) or with the complementary sense RNA as control
(C, D). Specific hybridization signals in the transmitting tract (A) and in the central tissue between the two seed-bearing structures
(B) are marked with arrows. Cross-sections of the pedicel were hybridized with PcCHI2 antisense RNA (E) or with the corresponding sense
RNA as control (F). Specific signals in the parenchymatic collenchyme are marked with arrows (E). a, anther; f, filament; fu, funiculus;
m, micropyle; s, seed; tt, transmitting tissue. Bar = 50 m.
 Chitinases from Parsley 673

Discussion monophyletic groups, most likely derived from a common

Sequence comparison at the cDNA and deduced protein
levels along with functional analysis identified both Pc-
CHI1 and PcCHI2 as class II endochitinases, despite their
large structural divergence and greatly differing expres-
sion modes. Phylogeny reconstruction revealed that the
sequence-related plant chitinases of classes I, II, and IV
form two major clusters (Figure 8), in agreement with a pre-
vious report (Hamel et al., 1997). The phylogram clearly
shows that class I and IV chitinases form well-separated
ancestor that predates the divergence of mono- and
dicotyledonous plants. In contrast, class II chitinases ex-
hibit a complex pattern of phylogenetic relationships.
They are dispersed among both class I and class IV chiti-
nases, indicating a polyphyletic origin, with independent
excision events eliminating the hevein domain at various
stages throughout the evolution of plant species. Interest-
ingly, the two parsley chitinases are evolutionarily well
separated not only from one another but also from the
majority of class II chitinases (PR-2 type, also referred to

Fig. 8 Phylogenetic Reconstruction Based on Amino Acid Sequences of Chitinases.

The most parsimonious tree was found using the TBR heuristic branch swapping algorithm with the PAUP 3.1.1 program. The chitin-bind-
ing protein agglutinin from stinging nettle (Urtica dioica) showing high sequence similarity but lacking chitinases activity (Lerner and
Raikhel, 1992) was used as outgroup to root the tree. The parsley chitinases PcCHI1 and PcCHI2 are highlighted in bold. Class II chiti-
nases, as defined by the absence of the hevein domain, are shaded. I*, class I chitinases lacking the C-terminal vacuolar targeting signal,
also referred to as class Ib (Collinge et al., 1993). According to the classification and nomenclature of plant chitinases given elsewhere
(Neuhaus, 1999), Urtica dioica agglutinin is referred to as class V, Beta vulgaris Ch1 as class VI, and the proteins of the class IV branch lack-
ing hevein domains (Triticum aestivum Cht2, Oryza sativa Chi2b, Citrus sinensis Chi1) as class VII chitinases. Database accession num-
bers are given on the right.
674 Y. Ponath et al.
as class IIa: Neuhaus, 1999) which are prevalent amongst
the Solanaceae (Capsicum, Lycopersicon, Nicotiana,
Petunia, Solanum).
PcCHI2 groups together with Chi2a from cotton
(Gossypium hirsutum), ChtSK2 from potato (Solanum
tuberosum), Chi2 from tomato (Lycopersicon esculen-
tum), and Chn134 from tobacco (Nicotiana tabacum).
ChtSK2 and Chi2, like PcCHI2, have been shown to be ex-
pressed in flowers (Wemmer et al., 1994; Harikrishna et al.,
1996) and Chn134 has been suggested to represent a
common evolutionary precursor of class I and class II
chitinases (Ohme-Takagi et al., 1998). The phylogram also
clearly indicates that the group containing PcCHI2 is more
closely related to the various class I chitinases than to the
PcCHI1-containing group of class II chitinases. PcCHI1 is
most similar to class II chitinases from peanut (Arachis
hypogaea) and Bermuda grass (Cynodon dactylon) and
together they form a sister group to the class I chitinases,
indicating an early evolutionary separation. Their closest
relatives are chitinases of class I (with and without C-ter-
minal extension) from monocotyledonous plants, as well
as several class IV chitinases. Hence, rather than confirm-
ing the previous definition of classes, the mingling of class
II chitinases with those from classes I and IV, as revealed in
Figure 8, seems to indicate that class II represents an arti-
ficially designated group of plant chitinases with no func-
tional or evolutionary bearing. Apparently, deletion of the
hevein domain occurred independently at various stages
through evolution and its presence or absence from chiti-
nases thus is a poor criterion for classification.
Despite their structural divergence, all chitinases share
strictly conserved sequence elements, including the pro-
posed catalytically active glutamic acid residue and sev-
eral other amino acids that are located in similar relative
positions (Holm and Sander, 1994; Iseli-Gamboni et al.,
1998), as indicated in Figure 1. Another characteristic
structural feature is an N-terminal putative signal peptide
whose occurrence in PcCHI1 and PcCHI2 (Figure 1),
together with the apparent absence of C-terminal vacuo-
lar targeting sequences (Neuhaus et al., 1991b), would
be in agreement with their excretion into the culture me-
dium of elicitor-stimulated parsley cells, where most of
the induced chitinase activity was found (Kirsch et al.,
1993).
Glycosylated chitinases have been isolated from a vari-
ety of plants (de Jong et al., 1992; Nielsen et al., 1994;
Ancillo et al., 1999), including parsley (Kirsch et al., 1993),
and the occurrence of potential glycosylation sites as well
as apparent size differences between the plant- and
E. coli-derived proteins suggest that PcCHI1 is also glyco-
sylated. However, the functional relevance of chitinase
glycosylation is unclear, and the non-glycosylated form of
PcCHI1 exhibited efficient chitinase activity. Generally,
glycosylation is thought to play a role in protein stabiliza-
tion and subcellular localization, which may apply to
PcCHI1 as well.
While functional identification in vitro has been reported
for several class I chitinases (Allona et al., 1996; Pan et al.,
1996; Yun et al., 1996), this study represents an example
for class II chitinases. However, the demonstration of
chitin degrading activity in vitro, often but not always as-
sociated with lysozyme activity, is but a formal criterion for
classification of the enzyme as chitinase, with no precision
as to the metabolic function in vivo. In particular, the
almost ubiquitous occurrence of at least one of multiple
chitinase isoforms in many tissues throughout many de-
velopmental stages of all plant species analyzed suggests
an important but unidentified endogenous role during
plant development. By contrast, an exogenous function
as an inducible protective agent against pathogens is eas-
ier to predict, e. g., from our present results. PcCHI1 was
induced strongly, rapidly, transiently and locally around
infection sites, concomitant with many other enzymes and
mRNAs associated with the early, local defense response
(Schmelzer et al., 1989; Somssich et al., 1989; Kawalleck
et al., 1995; Reinold and Hahlbrock, 1996; Batz et al.,
1998), whereas PcCHI2 was induced slowly and systemi-
cally with more or less equal distribution throughout the
infected leaf or even the whole organism. Thus, in agree-
ment with the behavior in elicitor-stimulated cell cultures,
the two parsley chitinases conform to the two classical
modes of pathogen defense, PcCHI1 to the immediate
and short-term local defense response and PcCHI2 to the
relatively long-lasting systemic acquired resistance (Kom-
brink and Somssich, 1997). As far as we are aware, this is
the first demonstration of such differential behavior of
chitinase isoforms within the same plant species.
Antimicrobial activity of chitinases, alone or in combi-
nation with glucanase, has been amply demonstrated
either in vitro or in transgenic plants (Zhu et al., 1994; Jach
et al., 1995; Jongedijk et al., 1995; Kombrink and
Somssich, 1997). However, target organisms are most
probably only fungi with chitin-containing cell walls, or
bacteria as far as the chitinases have lysozyme activity.
The parsley chitinases described here are therefore un-
likely to participate directly in the defense against P. sojae,
a chitin-less oomycete. This apparent discrepancy may
indicate a rather stereotyped plant response to any kind of
infection, involving all potential defense devices that can
be mobilized under a given circumstance, irrespective of
the precise nature of the pathogen. Such general type of
response would have the advantage of ensuring the best
possible defense even in those cases where evolutionary
adaptation of the two organisms has not occurred (Kom-
brink and Somssich, 1995).
Although chitin from fungal cell walls may be one or
even the major substrate for elicitor- or pathogen-induced
plant chitinases, their precise mechanism of action in
disease resistance is unknown. In addition to, or instead
of, impairing the pathogen by degrading its cell wall, chiti-
nases may liberate mono- or oligomeric fragments with
elicitor or other signaling properties to induce or potenti-
ate the defense response. Circumstantial evidence in this
direction has been obtained in several independent stud-
ies (Roby et al., 1987; Kurosaki et al., 1988; Ren and West,
1992).

More concrete data have been reported on putative en-
dogenous functions of plant chitinases in conjunction with
the hormone-like action of lipo-chitooligosaccharides.
These compounds, which may be derived from exoge-
nous (Long, 1996) as well as endogenous sources (Ben-
hamou and Asselin, 1989), not only have an important role
in root nodule formation in leguminous plants (Long,
1996), but have also been shown to rescue a growth-
arrested carrot (Daucus carota) somatic embryo mutant
(de Jong et al., 1993). In these connections, endochitinas-
es have been suggested to both generate these signal
molecules from GlcNAc-containing cell-wall components
(de Jong et al., 1993) and subsequently degrade and thus
inactivate them (Staehelin et al., 1994a, b). Preliminary ex-
periments indicated that PcCHI1 and PcCHI2 hydrolyze
certain lipo-chitooligosaccharides in vitro (Ponath, 1997),
raising the possibility that their metabolic functions are
similarly associated with the regulation of such putative
signal molecules, perhaps including the previously ob-
served down-regulation of cell cycle-related activities at
infection sites (Logemann et al., 1995).
Clarification of these questions and possibilities will be
a major task for future research on the precise role of these
and other chitinases. The striking differences observed
here at all levels analyzed, from various structural features
to the endogenously and exogenously regulated expres-
sion modes, suggest distinct, probably multiple physio-
logical roles for PcCHI1 and PcCHI2. As for all other chiti-
nases, their common classification may be regarded
merely as a tentative, formal assignment with little bearing
on the precise metabolic function. Our present results may
provide a suitable basis for further investigations in this
direction.
Materials and Methods
Cell Culture and Plant Material
Cell suspension cultures of parsley (Petroselinum crispum) were
propagated for 6 days as previously described (Kombrink and
Hahlbrock, 1986) and treated either with the synthetic Pep25
oligopeptide elicitor (Nürnberger et al., 1994) or with the equiva-
lent amount of water as a control. Cells were harvested at the
indicated times, frozen in liquid nitrogen, and stored at – 80°C.
Parsley plants (P. crispum cv Hamburger Schnitt) were grown
from seeds and inoculated with zoospores of Phytophthora sojae
as described (Reinold and Hahlbrock, 1996).
DNA Cloning and Analysis
Two ZAPII cDNA libraries were constructed from mRNA of pars-
ley cells that had been treated with elicitor for 0.5 – 3 h and 20 h,
respectively. They were screened with a 5 EcoRI fragment of the
potato basic chitinase ChtB3 (Beerhues and Kombrink, 1994).
After plaque purification, in vivo excision of pBluescript SKII
Phagmids (Short and Sorge, 1992) was achieved using the ExAs-
sist/SOLR system. The longest cDNAs were sequenced by the
dideoxynucleotide chain-termination method using T7 DNA poly-
merase (Pharmacia, Freiburg, Germany). Suitable sequencing
primers were synthesized on a Model 380A DNA Synthesizer (Ap-
plied Biosystems, Foster City, USA). Sequences were analyzed
Chitinases from Parsley 675
with the GCG software package (Devereux et al., 1984). The se-
quences reported in this paper have been deposited in the Gen-
Bank database with accession nos. AF141373 (PcCHI1-1),
AF141374 (PcCHI1-2), AF141372 (PcCHI2).
Protein Expression and Purification
After removal of the putative N-terminal ER signal sequences (von
Heijne, 1983) by restriction digestion and PCR, the cDNAs were
cloned as BamHI/KpnI fragments into the Escherichia coli ex-
pression vector pQE30 (Qiagen, Hilden, Germany) and the
correct frame confirmed by sequence analysis. Proteins were
expressed using E. coli strain SG13009[pRP4] (Qiagen) as rec-
ommended by the supplier. E. coli cells were transferred to 500 ml
L-medium containing 100 g/ml ampicillin and 25 g/ml kana-
mycin and grown at 37 °C (OD600: 0.7– 0.9). Expression of the
heterologous proteins was induced by adding isopropyl-1-thio-
-D-galactoside (IPTG) to a final concentration of 1 mM. After in-
cubation at 37 °C for 3 h, the cells were harvested by centrifuga-
tion, the pellet was resuspended in 10 – 15 ml buffer A (50 mM
sodium phosphate, 300 mM NaCl, pH 7.8) and bacteria were son-
icated (10  20 sec with interruptions of 20 sec). The 6  His-
tagged proteins were renatured and purified on Ni-NTA agarose
(Qiagen) as described (Holzinger et al., 1996).
Chitinase activity of the heterologously expressed proteins
was measured using the radiometric assay (Kombrink et al.,
1988).
Antisera
The heterologously expressed proteins were purified from E. coli
extracts under denaturing conditions on Ni-NTA agarose (Qiagen)
as recommended by the manufacturer using 8 M urea in all buffers.
Rabbits were boostered six times with 100 g each of the recom-
binant proteins within 4 months (Eurogentec, Seraing, Belgium).
The antisera from the final bleeding were used.
RNA Blot Hybridization
Total RNA was isolated (Lois et al., 1989), denatured, separated
on 1.2% agarose-formaldehyde gels, transferred onto nylon
membrane (Hybond, Amersham-Buchler, Braunschweig, Ger-
many) using the conditions recommended by the manufacturer
and cross-linked under UV light. Labeling of DNA probes and hy-
bridization of the blots were performed as described (Schröder
et al., 1992), except that the temperature was increased to 60 °C
and formamide was not included in the hybridization solution.
Blots were washed three times in 0.2  SSC, 0.5% SDS at 60 °C.
Immunoblotting
Protein extracts were prepared (Kombrink et al., 1988) and the
protein concentrations determined according to Bradford (Brad-
ford, 1976) with bovine serum albumin (BSA) as standard. Dis-
continuous SDS-polyacrylamide gel electrophoresis, using 0.75-
mm slab gels containing 12.5% acrylamide, and immunoblotting
were carried out as previously described (Laemmli, 1970; Towbin
et al., 1979). Proteins were transferred electrophoretically to nitro-
cellulose sheets (BA85, Schleicher & Schuell, Dassel, Germany)
with transfer buffer consisting of 5 mM Tris/HCl, 200 mM glycine
and 20% (v/v) methanol. Following saturation overnight with
blocking solution (5% milk powder in PBS: 12.5 mM sodium phos-
phate, pH 7.5, 0.15 M NaCl), nitrocellulose sheets were incubated
with antiserum (anti-PcCHI1 at a dilution of 1:200, anti-PcCHI2 at
a dilution of 1:500) in blocking solution. Protein-antibody com-
plexes were detected using peroxidase-conjugated goat
anti(rabbit IgG) antibodies (Bio Genes, Berlin, Germany) and the
ECL detection system (Amersham, Braunschweig, Germany).
676 Y. Ponath et al.
Immunohistochemical Staining
The methods used for indirect immunohistochemical staining
were essentially the same as described by Reinold and Hahlbrock
(Reinold and Hahlbrock, 1996). For blocking nonspecific anti-
body binding, sections were incubated in 1% BSA/PBS. Anti-
PcCHI1 and anti-PcCHI2 antisera and alkaline phosphatase-
coupled anti-rabbit secondary antibodies were used at a dilution
of 1:100 in 1% BSA/PBS. The color reaction was performed with
nitroblue tetrazolium and bromochloroindolyl phosphate (Sigma-
Aldrich, Deisenhofen, Germany) according to the manufacturer’s
instructions.
In Situ RNA/RNA Hybridization
For in situ hybridization, the plant tissue was fixed and embedded
in paraffin (Reinold and Hahlbrock, 1996). Two series of adjacent
sections were prepared for hybridization to sense (control) and
antisense RNA probes which were generated from linearized
plasmids with T3 or T7 polymerase and uridine 5-[-35S]thio-
triphosphate (400 Ci/mmol –1; Amersham-Buchler, Braunschweig,
Germany).
Sequence Comparison
Published chitinase amino acid sequences, using those showing
less than 90% identity to each other, were aligned using the
PileUp program of the GCG program package, version 10.0
(Devereux et al., 1984) with the gap weight set to 6 and the gap
length weight set to 1. Based on this alignment, a maximum par-
simony analysis was performed (Fitch, 1977) using the PAUP
3.1.1 program (Smithsonian Institution, Washington DC, USA)
with a data matrix of 282 positions comprising the complete cat-
alytic domain (corresponding to amino acid positions 26 to 267 in
PcCHI2). The most parsimonious tree was found using the heuris-
tic search option with the ‘tree bisection reconnection’ (TBR)
branch swapping algorithm (Swoffort et al., 1996). For statistical
analysis, 100 bootstrap replications were analyzed (Felsenstein,
1985). Treating character weights as repeat counts resulted in
bootstrap values ranging from 85 to 100.
Acknowledgements
We are grateful to Elke Logemann and Drs. Olaf Batz, Edda Koop-
mann and Susanne Reinold for generous help and supply of
materials.
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