J. Plant Physiol. 158.

1463 1469 (2001)

Urban & Fischer Verlag
http://www.urbanfischer.de/journals/jpp

Expression of cysteine proteinase mRNA in chickpea (Cicer arietinum L.)

is localized to provascular cells in the developing root
Emilio Cervantes1 *, Juana G. De Diego2, Mara Dolores Gmez3, Javier De Las Rivas1, Jos Mariano Igual1,
Encarna Velzquez4, Philippe Grappin5, Manuel Cercs3, Juan Carbonell3
1

IRNA-CSIC, Apartado 257, 37079, Salamanca, Spain

Departamento de Bioqumica y Biologa Molecular, Edificio Departamental, Campus Miguel de Unamuno, Universidad de Salamanca, 37007,
Salamanca, Spain
3

Instituto de Biologa Molecular y Celular de las Plantas (IBMCP), CSIC-Universidad Politcnica de Valencia, Camino de Vera s/n, 46022, Valencia,
Spain
4

Departamento de Microbiologa, Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007, Salamanca, Spain

Laboratoire de Biologie de Semences, INRA, 78026, Versailles Cedex, France

Received April 5, 2001 Accepted July 2, 2001

Summary
A full-length cDNA clone encoding a chickpea (Cicer arietinum L.) cysteine proteinase, homologous
to rd21gene of Arabidopsis as well as to cysteine proteinase genes from legumes, was isolated.
Expression of the mRNA in growing radicles has been located in a very precisely defined longitudinal zone in the stele, corresponding to the area of differentiation of vascular tissues. This result
suggests that the encoded proteinase may be involved in programmed cell death events that precede vascular development.
Key words: Cicer arietinum cysteine proteinase germination root development vascular
bundle
Abbreviations: aa aminoacids. PCD Programmed Cell Death

Introduction
Germination and early seedling development are highly complex processes, directed by the coordinate action of multiple
regulatory pathways. Very early in root development, PCD
* E-mail corresponding author: ecervant@usal.es
The nucleotide sequence reported in this paper has been submitted
to the EMBL Nucleotide Sequence database under accession number X82011.

must occur in order to allow the co-ordinate formation of new

vascular tissue (Fukuda 1997, Ye and Varner 1996). Thus, in a
young seedling, a limited number of cell divisions separate
the quiescent center from the cells that are precursors to the
vascular bundle, and in a short space occupied by a reduced
number of cells, the proteolytic events that lead to cell death
must be accurately programmed.
Proteinases have been purified and their corresponding
cDNAs cloned from legume seeds and seedlings (Becker et
al. 1994, Jones et al. 1996, Cercs et al. 1999, Fischer et al.
0176-1617/01/158/111463 $ 15.00/0

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Emilio Cervantes et al.
Figure 1. Multiple alignment of the CacG1 sequence (364 aa) from Cicer arietinum together with 6 cysteine proteinases from plants: 3 similar proteinases from other legumes (Vicia sativa 368 aa,
Phaseolus vulgaris 364 aa, and Pisum sativum 367aa); 2 of the closest proteinase sequences from the Arabidopsis thaliana genome (ARATH2, 462 aa, that is rd21-arath from SWISSPROT database and ARATH1, 376 aa, with access-number 023185); and caricain from Carica papaya (348 aa). The last 90 aa of the ARATH2 sequence is not shown because of the lack of homology to the
rest of the alignment. Several domains predicted in the sequence of CacG1 are indicated: a signal peptide from aa 1 to 33; an activation peptide from aa 34 to 110; and the mature protein, from
aa 110 to 363, which includes an initial variable region of 20 aa. Moreover, two different domains inside CacG1 protein are recognised: an alpha helix domain from aa 142 to 235 and a mainly
beta domain from 131 to 141 and 236 to 364. The secondary structure prediction (H for alpha helix, E for beta strands, and L for loops), together with the solvent accessibility (b for buried zone
and e for external region), are also presented in the last two lines. Below the alignment, a drawing with the main secondary structure features gives a better view of the full, structural prediction.
In the CacG1 sequence, the fully conserved charged aa as well as six cysteines that form sulphide bridges (SH1, SH2 and SH3), are marked with a box.

Expression of a cysteine proteinase

2000 and references therein); however, their expression patterns and regulatory mechanisms have not been clarified.
The specific physiological role for each described proteinase
is also a matter of controversy, and their substrate proteins
remain to be identified.
In animals, the PCD programs consist of a cascade of proteolytic events, accurately regulated, in which a particular set
of cysteine proteinases, the caspases, have a predominant
role (Cryns and Yuan 1998). In plants, cysteine proteinases
have been associated with PCD processes (Fukuda 1997),
and the existence of caspases has been suggested, based
mainly on experiments that used inhibitors (Del Pozo and
Lam 1998), but so far, no sequences with homology to caspases have been identified.
In this work, we describe the cloning, expression pattern,
and structural analysis of a cysteine proteinase gene, cacG1,
that is induced during chickpea germination. In situ hybridization experiments show mRNA expression in a precise zone
corresponding to vascular element differentiation in the developing root. That the cacG1 gene is expressed in cells associated with the maturation of vascular tissue, both in xylem
and phloem precursor cells, suggests involvement in the
control of protein degradation during this process.

Materials and Methods

Plant material
Seeds of chickpea (Cicer arietinum, L.) were imbibed on moistened
filter paper and germinated in the dark at 25 C. For the treatments
with ethylene, the excised cotyledons were imbibed on filter paper in
closed, 2-L tupper-waretm containers (20 seeds per container). Ethylene was injected through a rubber stopper adapted to the lid.

1465

Radio labeling of probes

For the northern blots, the complete insert of clone cacG1 was purified from agarose gels with Geneclean (BIO 101 inc.). DNA probes
were radio labeled with [32P]dCTP using random primers according
to Feinberg and Vogelstein (1983).

Nucleotide sequencing
Nucleotide sequences were obtained using double stranded plasmid
template DNA and the T7 nucleotide sequencing kit from Pharmacia.

Protein structure analysis

Proteins with a significant similarity to the CacG1 deduced amino
acid sequence were found in the EMBL/SWISSPROT databases,
using the methods FASTA (Pearson and Lipman 1988) and BLAST
(basic local alignment search tool) (Altschul et al. 1990) as search
tools. A multiple alignment of 16 sequences of homologous proteins
from plants was constructed using the CLUSTAL-X program (Thompson et al. 1997). The alignment allows the identification of fully conserved residues and the recognition of the amino acids involved in disulphide bridges and in the active site. A tree of similarity, deduced
from pair-wise comparison and cluster analysis of the sequences,
was also constructed to better evaluate the similarities of the sequences. The alignment and the consensus sequence derived were
used for secondary structure and solvent accessibility predictions by
the PHD method (Rost et al. 1994). The comparison with the 3Dstructure of procaricain (1pciA in PDB database) and papain (1ppn in
PDB database) allowed the recognition of the domains of the CacG1
protein.

In situ hybridization
In situ hybridization experiments were done, essentially as described
by Davies (1993), using digoxigenin labeled RNA probes. For tissue
preparation, radicles of germinated chickpea seedlings (48 hours
post imbibition, 2 cm in length) were excised and divided into five
equal portions (see Fig. 3). Paraffin sections (7 m) corresponding

RNA isolation and northern blot analysis

Seeds (or excised cotyledons where indicated) were ground in liquid
nitrogen and RNA isolated by the method of Wadsworth et al. (1988).
Northern blots were prepared as described by John et al. (1995). The
gels were stained with ethidium bromide and the rRNA bands visualized to verify equal sample loading.

cDNA library construction and screening

Poly (A) + RNA was purified by affinity chromatography using an oligo-dT cellulose column (Boehringer). Double stranded cDNA was
synthesized using poly (A) + RNA isolated from germinated seeds
using a Zap-cDNA synthesis kit (Stratagene). The cDNA library was
constructed in Lambda Zap II vector (Stratagene) according to the
manufacturers instructions. To clone cacG1 cDNA, the library was
screened with the PCR fragment encoding the partial sequence for a
putative cysteine proteinase (Cervantes et al. 1994).

Figure 2. Northern blot showing cacG1 transcript expression in the

excised cotyledons (imbibed after ablation of the embryonic axis)
under different ethylene concentrations. All the samples were collected at 48 hours after imbibition. Control corresponds to normal cotyledons from intact seedlings.

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Emilio Cervantes et al.

to portions 1 and 2 were employed in the experiments. Sense and

antisense RNA probes were generated using a standard protocol for
in vitro transcription, with the Dig RNA labeling mix (Boehringer Mannheim) as source for the nucleotides. Templates were obtained by digestion of the plasmids with EcoR1 or Bsp120, respectively, for the
sense and antisense RNA probes.

Results
Cloning and sequencing of cacG1
Screening of the cDNA library made from cotyledons of germinated chickpea seedlings, with the radio labeled PCR fragment corresponding to a putative cysteine proteinase (Cervantes et al. 1994; accession number X70375), yielded several positive clones. The corresponding phagemids in vivo
were excised and the complete nucleotide sequence of one
of the clones was determined. The clone was named cacG1:
it is 1217 nucleotide pairs in length, and it contains one open
reading frame (1092 nucleotides) flanked by 5 and 3 noncoding sequences and ends in a poly-A tail. This sequence
contains a region with > 95 % identity to the PCR fragment
that was used a s a probe.

tivum,); two of the closest proteinase sequences from the

Arabidopsis thaliana genome; and the mentioned Caricain
from Carica papaya. Finally, Figure 1 also shows other structural features that can be distinguished in CacG1. The three
amino acids that constitute the active site of this papain-type
of cysteine proteinases are fully conserved in the alignment,
illustrated by a grey background in the figure. These residues
in CacG1 sequence are: Cys155, His311, and Asn331. Six
fully conserved cysteine residues that form three sulphide
bridges in the proteinase structure are also recognized and
marked in Figure 1. All these features allow a good structural
assigment of the CacG1 sequence, and support a cysteine
proteinase activity for CacG1.

Expression of cacG1 in seeds during germination

cacG1 mRNA was undetected in dry seeds as well as in
seeds imbibed for 2 and 6 hours. The signal was first detected at 12 hours after imbibition and increased, reaching a
maximum at 4 days (Cervantes et al. 1994 and unpublished
results).

Structural features of cacG1

cacG1 encodes a protein of 364 amino acids. Comparative
analysis of this sequence showed characteristics of an endopeptidase of the papain superfamily. A complete search in
the protein databases was done in order to find out the most
similar protein sequences of other plant proteases. Based on
the similarity, 16 sequences of cysteine proteinases were selected and a multiple sequence alignment was done. This allowed the identification of three different regions in the sequence of CacG1 protein: a signal peptide from aa 1 to 33,
with characteristics of a leader sequence; a propeptide from
aa 34 to 110, which might block the active site; and the mature protein, from aa 110 to 364, which is the active enzyme
and includes an initial 20 aa variable region, where the cleavage for activation occurs. Figure 1 shows the sequence of
CacG1 with the structural features described above. The
strong homology with caricain, a proteinase of known 3D
structure, allows a recognition of two different domains inside
CacG1 protein: an alpha helix domain from aa 142 to 235,
and a mainly beta domain from 131 to 141 and 236 to 364.
The recognition of these domains is also obtained by an
independent prediction of secondary structure of the sequence (Rost et al. 1994). Figure 1 illustrates this secondary
structure prediction, together with the solvent accessibility of
the different parts of the sequence. In the figure, the sequence of CacG1 is called as CacG1 364 (as being a Cicer
arietinum protein with 364 aa), and it is aligned to 6 sequences: three of very similar cysteine proteinases of other
legumes (Vicia sativa, Phaseolus vulgaris and Pisum sa-

Figure 3. Outline of the in situ hybridization experiment. Germinated

chickpea seedlings were selected at a length of 2 cm and divided
into five equal portions of 4 mm each. Portions 1 and 2 were used for
the in situ hybridization experiments.

Expression of a cysteine proteinase

1467

Figure 4. A to E: In situ hybridization experiment: B and D: Hybridization with the antisense cacG1 probe in one of the microtome sections of the
upper part of portion 1 showing an intense mRNA expression in the central cylinder in the cells precursor of xylem and phloem. A and C: Control
hybridized with the sense probe in the same region as B and D. E: Hybridization with the antisense cacG1 probe in one of the sections of the
lower part of portion 2 showing only a faint positive signal. F: Safranin staining in a section close to E. Bar equals 100 mol/L.

1468

Emilio Cervantes et al.

Expression of cacG1 mRNA in excised cotyledons is

responsive to ethylene
Figure 2 shows the effect of different ethylene concentrations
on the levels of cacG1 RNA in excised cotyledons (cotyledons imbibed after ablation of the radicle). The control lane
shows transcript levels in cotyledons of intact seeds at 48
hours after imbibition. The results show that ethylene gas at a
concentration of 100 L/L was able to increase cacG1 transcript levels in excised cotyledons. This induction is independent of the pH because the same results were obtained
at pH 3.2 and pH 7.0 (not shown). Dose-response analysis of
this effect is comparable to other ethylene-regulated genes
that show maximal induction at around 100 ppm ethylene
(Chen and Bleecker 1995).

Expression of cacG1 associated with vascular tissue

differentiation in developing radicles
As ethylene was able to induce the expression of cacG1
mRNA, and since this phytohormone has been reported to
be involved in the programmed cell death (PCD) in plants
(Orzaez and Granell 1997), we decided to investigate cacG1
cysteine proteinase mRNA levels in the early development of
vascular tissue in the radicle. Thus, 2-cm-length radicles of
chickpea seedlings at 48 hours post-imbibition were selected
and divided longitudinally into 5 portions (4 mm each, see
Fig. 3). Sections corresponding to portions 1 and 2 in Figure
3 were prepared for the in situ hybridization experiments.
cacG1 mRNA was only detected throughout a region located
between 3 and 4 mm from the root apex, i.e. always in portion
1, and thus corresponding to early stages of vascular element differentiation (Fig. 4 B, D). Samples prepared from portion 1 were also hybridized, with the sense probe giving no
signal (Figs. 4 A, C). Expression of cacG1 mRNA was not detected in the stele of sections corresponding to portions 2
through 5 in the radicle. Figure 4 E shows antisense staining
in a section close to where expression occurs, i.e. lower sections of portion 2 showing only a faint signal in the periphery
of the stele.

Discussion
In a previous work a partial cysteine proteinase cDNA was
amplified by PCR (Cervantes et al. 1994). Expression analysis showed that the corresponding gene was upregulated
during germination of chickpea, and ethephon induction suggested that ethylene was involved in the process. In this
work, we used that cDNA to screen a cDNA library from germinated chickpea seeds and to clone the corresponding
cDNA. A full-length cDNA with conserved features of plant
cysteine proteinases was obtained and used as a probe in

northern hybridizations. The corresponding transcript is undetected in dry seeds and detected for the first time between
6 and 12 hours after seed imbibition. Our results with ethylene gas indicate that the reported ethephon induction in excised cotyledons is due to ethylene and not to any other
chemical released by ethephon. Thus, excised cotyledons
respond to ethylene and cacG1 expression is ethylene sensitive, with a dose-response relationship similar to other ethylene mediated responses (Chen and Bleecker 1995).
Results on the localization of cacG1 transcript by in situ
hybridization in growing radicles after imbibition showed a
very well-defined expression in an area that corresponds to
cells that will give rise to the vascular bundle, in both tracheary elements as well as phloem cells (Mitler and Lam
1995). Darker cells are, in general, smaller and may correspond to an earlier differentiation stage. The transcript was
not detected in the root in regions located above and below
this area (Fig. 3). Based on these observations, we may
speculate that the cysteine proteinase encoded by cacG1
could be involved in the metabolic events corresponding to
the early steps that lead to cell death in this region, and that
these early steps may be common between xylem tracheary
elements that undergo autolysis and PCD and phloem cells
that remain living after differentiation. Other cysteine proteinases have been reported to be associated with processes of
PCD in plants (Ye and Varner 1996, Beers 1997, Fukuda 1997,
Xu and Chye 1999). These plant proteinases do not belong to
the family of caspases identified in animal systems (Cryns
and Yuan 1998). To date, only indirect evidence based on inhibitor studies suggests that caspases may be active in PCD
processes in plants (Del Pozo and Lam 1998); thus, in plants,
other cysteine proteinases of the papain superfamily may be
important in this process. More biochemical evidence will be
required to confirm the precise roles of the different proteinases.
One interesting aspect may concern the possible role
played by ethylene in the regulation of the expression of
cacG1 mRNA. Ethylene has been reported to be involved in
many processes related to senescence and aging in plants
(Orzaez and Granell 1997, Nooden and Leopold 1988), as
well as in the PCD processes that lead to vascular formation
(Ye and Varner 1996) and aerenchyma formation mediated
by hypoxia (He et al. 1996). On the other hand, ethylene is involved in the induction of a -glucanase gene in embryonic
axes of germinated pea seedlings after 50 hours of imbibition
(Petruzelli et al. 1999). Further analysis of mechanisms regulating gene expression during seed germination may reveal
new aspects about the complex role of ethylene in plant development.

Acknowledgements. We thank Pilar Colorado and Antonio Rodrguez

for help with the cDNA library construction. Mara del Mar Snchez
helped with the nucleotide sequencing analysis.

Expression of a cysteine proteinase

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