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First published online June 19, 2003; 10.1104/pp.102.019562 Plant Physiology 132:1707-1715 (2003) © 2003 American Society of Plant Biologists Identification and Differential Expression of Two Thioredoxin h Isoforms in Germinating Seeds from Pea1Unité Mixte de Recherche 1191 Physiologie Moléculaire des Semences, Institut National de Recherche Agronomique - Institut National d'Horticulture - Université d'Angers, ARES, 16 Boulevard Lavoisier, 49045 Angers cedex 1, France
The NADPH/NADP-thioredoxin (Trx) reductase (NTR)/Trx system (NTS) is a redox system that plays a posttranslational regulatory role by reducing protein targets involved in crucial cellular processes in microorganisms and animals. In plants, the system includes several h type Trx isoforms and has been shown to intervene in reserve mobilization during early seedling growth of cereals. To determine whether NTS was operational during germination of legume seeds and which Trx h isoforms could be implicated, Trx h isoforms expression was monitored in germinating pea (Pisum sativum cv Baccara) seeds, together with the amount of NTR and NADPH. Two new isoforms were identified: Trx h3, similar to the two isoforms already described in pea but not expressed in seeds; and the more divergent isoform, Trx h4. Active recombinant proteins were produced in Escherichia coli and used to raise specific antibodies. The expression of new isoforms was analyzed at both mRNA and protein levels. The lack of correlation between mRNA and protein abundances suggests the occurrence of posttranscriptional regulation. Trx h3 protein amount remained constant in both axes and cotyledons of dry and imbibed seeds but then decreased 2 d after radicle protrusion. In contrast, Trx h4 was only expressed in axes of dry and imbibed seeds but not in germinated seeds or in seedlings, therefore appearing as closely linked to germination. The presence of NTR and NADPH in seeds suggests that NTS could be functional during germination. The possible role of Trx h3 and h4 in this context is discussed.
Thioredoxins (Trxs) constitute a family of small and ubiquitous proteins with two close and active Cys residues in a conserved motif: WCG/PPC. In their dithiol form, they are powerful disulfide reductases (Holmgren, 1985  ) that play a
posttranslational regulatory role on protein targets involved in an
ever-increasing number of cellular processes.
Plant cells have four different types of Trx isoforms, the well-known f and
m types in the chloroplast (Buchanan,
1991 To find out whether the NTS was operational during germination of legume seeds, we characterized the expression of Trx h isoforms in germinating pea (Pisum sativum) seeds and brought to light two new isoforms. We also determined the relative amounts of NTR and the content in NADPH, the two components of the system that drive Trx h isoform reduction. Expression was investigated separately in cotyledons that mainly serve to reserve breakdown and in embryonic axes that prepare for cell elongation and cell division associated with radicle emergence. In this paper, we report for the first time, to our knowledge, about a NTS in germinating pea seeds that involves two new Trx h isoforms that are differentially expressed at protein levels.
Trx h Isoforms Expressed in Seeds
As a preliminary to the Trx h expression studies during germination, we
established the germination rate and water uptake of pea seeds
(Fig. 1). When pea seeds were
imbibed at 20°C in the dark, 50% of the seeds had germinated after 25 h of
imbibition. As expected, the water uptake curve shows the typical triphasic
shape (Bewley, 1997
Two different isoforms of Trx h (GenBank accession nos. AJ319808 and AJ310990) have been identified previously in pea. Because both genes were given the same name, they will be referred to as Trx h1 and Trx h2. To investigate whether both genes were expressed in seeds, reverse transcription (RT)-PCR was performed using specific primers (Table I), using as templates cDNAs from dry or 22-h-imbibed seeds and cDNAs from 7-d-old green leaves. Figure 2 shows that Trx h1 and h2 were easily amplified in leaves but could not be detected in seeds even after 35 cycles of amplification. Cloning and sequencing of the PCR products amplified from leaf cDNAs confirmed that they actually encoded Trx h1 and h2, allowing the further use of these probes in northern experiments.
The lack of expression of these Trx h isoforms in pea seeds led us to
investigate whether other unknown isoforms could be specifically expressed
during germination. The presence of two conserved motives (WCG/PPC and
Q/EAMPTF) in most plant Trx h allowed the design of degenerated
oligonucleotide primers for RT-PCR experiments performed on cDNAs from
22-h-imbibed seeds (Table I).
Two forward primers, Xs-1A and Xs-1B, were derived from the WCG/PPC sequence
to take into account the presence of either a G (Xs-1A) or a P (Xs-1B) in the
third position. Similarly, two reverse primers, Xa-anti2A and Xa-anti2B, were
designed in the Q/EAMPTF consensus sequence, respectively. Of the four primer
combinations, only those using forward primer Xs-1A and reverse primers
Xa-anti2A or Xa-anti2B yielded products with an expected size of 170 bp (data
not shown). Cloning and sequencing of the PCR products revealed two new Trx h
cDNAs that will be referred to as Trx h3 and Trx h4. Among
all the clones analyzed, Trx h3 and h4 were encountered
several times, suggesting that they correspond to major transcripts of Trx h
in germinating seeds. The full-length sequences of h3 and h4
cDNAs (647 and 533 bp) were obtained by RACE and found to encode proteins of
113 and 130 amino acids, respectively, with theoretical masses of 12,578 and
14,549 D (GenBank accession nos. AY170650 and AY170651). The protein sequences
deduced from Trx h3 and h4 were aligned with those of Trx h1 and h2
(Fig. 3A). The sequence of Trx
h3 appeared similar to those of h1 and h2 (72% and 64% identity,
respectively), whereas that of Trx h4 was more divergent because it shared
only 37% to 41% identity with the three other isoforms. In addition, Trx h4
exhibits an N-terminal 17-amino acid extension enriched in hydrophobic
residues (Fig. 3A). A
comparison of the four pea sequences with all the Trx h sequences in databases
revealed that they were more similar to those of Arabidopsis than to any other
species (data not shown). Thus, a phylogenetic tree was constructed with pea
and Arabidopsis sequences (Fig.
3B). Pea Trx h1, h2, and h3 were found in the same subgroup as
Arabidopsis Trx h1, whereas Trx h4 was clustered in another subgroup that
included Trx h2 from Arabidopsis. This last cluster comprised so far only four
isoforms: Trx h2 from Arabidopsis, Trx h1 and h2 from soybean (Glycine
max; Shi and Bhattacharyya,
1996
To investigate whether Trx h3 and Trx h4 encoded functional proteins, cDNAs were cloned into the expression vector pASK-IBA3 designed to produce recombinant proteins with a C-terminal Strep-tag. Recombinant Trx h3st and Trx h4st were found to be highly expressed in E. coli as soluble proteins and were purified using Strep-Tactin-Sepharose (Fig. 4A). The purified recombinant proteins were used to raise polyclonal antibodies in rabbits. The specificity of the two antisera (anti-Trx h3st and antiTrx h4st) was demonstrated by the absence of cross reactions using the recombinant proteins as antigens (Fig. 4B). Both purified Trx h3st and Trx h4st readily reduced insulin with similar kinetics. They appear to promote a faster insulin reduction than the E. coli Trx used as a control (Fig. 5).
The expression of the four Trx h isoforms during germination and early
seedling growth of pea was first investigated by northern-blot analysis using
DNA probes derived from the cDNA coding regions. Because cross-hybridization
could occur between the different isoforms
(Brugidou et al., 1993 Transcripts of Trx h1 and h2 were not detected by northern blot in germinating seeds (Fig. 6), which confirmed the lack of detection by RT-PCR (Fig. 2). Thus, the signals obtained with the h3 probe in germinating seeds cannot be attributed to h1. Among the other organs tested during early seedling growth, h1 and h2 were only found in green leaves, albeit in a very low amount (Fig. 6). In contrast, Trx h3 and h4 were clearly detected in dry and imbibed seeds and in all other organs examined (Fig. 6). In dry seeds, Trx h3 and h4 transcripts were present both in embryo axes and cotyledons, transcript levels of h3 being higher than that of h4. Trx h3 and h4 were found to be differentially expressed during germination. In the course of imbibition, the content in h3 gradually decreased in cotyledons but increased in embryo axes before the onset of radicle protrusion (22 h) and afterward (Fig. 6). However, because a slight increase (600–650 b) in the size of detected band was systematically observed between early germination (0, 6, and 12 h) and latter stages (22 and 46 h), including 7-d-old seedlings, the presence of two very similar transcripts cannot be excluded. Trx h4 (550 b) amount started to increase during phase II of imbibition in both embryo axes and cotyledons (Fig. 6) and remained high during early seedling growth.
A further analysis of Trx h isoform expression was performed by quantitative RT-PCR. The lack of expression of Trx h1 and h2 during germination was further confirmed (data not shown). As for the northern blot, Trx h3 and h4 were found to be expressed in dry and imbibed seeds and in green leaves and roots (Fig. 7). In contrast, they showed similar expression profiles upon imbibition, with a strong induction of expression in axes after the radicle protrusion (46 h) and in cotyledons just before and after radicle protrusion (22 and 46 h). Concerning the isoform h4, this pattern is in accordance with that obtained in northern blot. Considering the expression of Trx h3 transcripts in cotyledons at early stages of germination, quantitative RT-PCR yielded different results because the amount of transcript was much lower than expected from the northern-blot signal (Figs. 6 and 7). Such a result might be explained by the presence of another cross-hybridizing isoform.
To investigate the expression of the protein isoforms Trx h3 and h4 in seeds during germination and early seedling growth, western blots were performed upon soluble proteins using the specific anti-Trx h3st and anti-Trx h4st antibodies (Fig. 8). Trx h4 exhibited an apparent higher molecular mass (14.5 kD) than Trx h3 (13 kD), which agrees well with the different theoretical molecular masses (14.549 and 12.578 kD, respectively) deduced from the cDNAs. Trx h3 was easily detected from 0 to 46 h of imbibition, both in cotyledons and embryo axes (Fig. 8). Although its level remained constant in both parts until 46 h, Trx h3 could not be detected anymore in cotyledons at 72 h, and its amount was markedly reduced in embryo axes at 72 h. It could be detected, albeit at low levels, in 7-d-old seedlings root tissues, but not in leaves (Fig. 8). The expression profile of Trx h4 was clearly different because the protein was only detected in embryo axes of dry or imbibed but ungerminated seeds (6–22 h), its amount decreasing slightly upon imbibition. The protein could not be detected in embryo axes and cotyledons of germinated seeds (46 and 72 h) or in the organs from 7-d-old seedlings examined. Thus, Trx h4 could be a seed-specific isoform.
A functional NTS requires the presence of the two other components of the system, namely NTR and the co-enzyme NADPH. The amount in NTR in embryo axes and cotyledons was investigated by western blotting using antibodies raised against Arabidopsis NTR, which revealed a single polypeptide with the expected size of 35 kD. NTR, which was detected in both tissues, was more abundant in embryo axes than in cotyledons. Its content slightly increased in axes after radicle protrusion, whereas it remained almost constant in cotyledons (Fig. 8).
Because NADPH is an essential metabolite undoubtedly required for
germination, we effectively measured NADPH amounts of 2 nmol
g–1 dry weight in germinating seeds, which agrees
well with other values reported in seeds and seedlings
(Zhao et al., 1987
Because the NTS is known to intervene in redox regulation of crucial molecular events in microorganisms and animal cells, similar functions in plant cells may be envisioned. The system has been extensively studied in germinated cereal seeds where its role in the mobilization of reserves was clearly established (for review, see Besse and Buchanan, 1997 ). We have addressed whether the NTS could be
functional in legume seeds by monitoring the expression of Trx h isoforms and
NTR during early germination and seedling growth of pea seeds. All three components of the system, NADPH, NTR, and Trx h, were demonstrated to be present in dry pea seeds and in germinating seeds, indicating a role of the system during germination. Trx h being encoded by multiple genes in plants, we have dissected the system by analyzing the expression of Trx h isoforms in germinating seeds. Because the transcripts of already known h1 and h2 isoforms were not detected, we designed a strategy to clone other putative isoforms that could be expressed in seeds. This approach revealed that two new isoforms, Trx h3 and h4, were expressed both at mRNA and protein levels during germination.
The two new isoforms, h3 and h4, exhibit a primary structure highly similar
with other Trx h and with the canonical disulfide active site (WCGPC). The
analysis of the pea sequences indicated that Trx h1, h2, and h3, which were
clustered together, differed more from Trx h4 than from Arabidopsis Trx h1.
Such an observation is consistent with the hypothesis that the apparition of
the numerous h isoforms had started before higher plant divergence
(Sahrawy et al., 1996
In contrast, Trx h4 belongs to the subgroup that includes Trx h2 from
Arabidopsis and Trx h1 and h2 from soybean
(Shi and Bhattacharyya, 1996
At the mRNA level, among the four known pea Trx h, only Trx
h3 and h4 were found to be expressed in dry and germinating
seeds, but also in seedlings. No transcript encoding Trx h1 and h2 could be
detected, suggesting these isoforms do not intervene in germination. In
addition, they were only slightly detected in green leaf tissue. The only
clues regarding their function consist in their annotations in GenBank
(accession nos. AJ319808 and AJ310990), with h2 being linked to oxidative
stress and development. Because Trx h3 and h4 were the major
Trx h in seeds, we produced recombinant proteins that proved to be active in
terms of insulin reduction, thus confirming the Trx activity of the isoforms.
We were then able to raise antibodies that were highly specific and, thus,
allowed to discriminate Trx h3 and Trx h4 expression at the protein level. It
is noteworthy that antibodies previously raised against Trx h generally
cross-reacted with several isoforms
(Lozano et al., 1996
Both Trx h3 and h4 were found in dry seeds either only in embryo axes (h4)
or in both axes and cotyledons (h3), indicating that they were synthesized
before germination. During germination, Trx h3 amount remained constant both
in embryo axes and cotyledons, whereas h4 was found only in axes, its level
decreasing slightly upon imbibition and the protein disappearing after radicle
protrusion. After germination, Trx h3 amount decreased slowly but was still
detected in 7-d-old seedlings. A decrease in the overall Trx h content was
also reported in cereals during germination and early seedling growth
(Lozano et al., 1996
The main result inferred from this work is the expression in seeds of two
new isoforms Trx h3 and h4, which raises the question of their functions. The
growing body of evidence that Trx h are involved in reserve mobilization in
cereals (Besse and Buchanan,
1997
The results presented here provide the first characterization, to our
knowledge, of the NTS in legume seeds. The system, which appeared operational,
involves new Trx h isoforms differentially expressed in seeds and, thus,
expected to carry different roles during germination. Further understanding of
the function of the system with regard to germination of legume seeds will be
addressed through the identification of Trx h isoforms targets using mutated
proteins as ligands in affinity chromatography or as baits in yeast
double-hybrid screening (Verdoucq et al.,
1999
Materials Pea (Pisum sativum L. cv Baccara) seeds harvested in 2000 were obtained locally from an agronomical research institute (Fédération Nationale des Agriculteurs Multiplicateurs de Semences, Brain-sur-l'Authion, France) and stored in sealed plastic bags at 5°C (70% relative humidity). Mature seeds were allowed to germinate on folded filter paper moistened with water (0.18 mL cm2) at 20°C in the dark or in the light (16 h of light/8 h of dark). Water content was determined by measuring the fresh and dry weight of the seeds (the latter was determined after heating at 100°C for 3 d).
Total RNAs were extracted according to Verwoerd et al.
(1989 The two already known sequences of pea Trx h cDNAs (GenBank accession nos. AJ319808 and AJ310990, respectively, numbered h1 and h2 for convenience in this paper) were amplified by RT-PCR using specific primers designed at the 5' and 3' ends of their coding sequence (Table I). To amplify cDNAs from Trx h that could be expressed during seed germination, four degenerated primers (Xs-1A, Xs-1B, Xa-anti2A, and Xa-anti2B) were derived from two motifs conserved in protein sequences of Trx h from different sources. PCR products were then cloned into the PCR4-TOPO vector of the TOPO TA Cloning Kit for Sequencing (Invitrogen) according to the manufacturer's instructions. To obtain the corresponding full-length cDNAs, 3'1, 3'2, 5'1, and 5'2 primers (Table I) were designed to perform RACE according to the GeneRacer kit's instructions. DNA sequencing was performed by MWG-Biotech AG (Ebersberg, Germany).
For quantitative RT-PCR experiments, cDNAs—obtained from 2 µg of
total RNAs by classical RT (Huang et al.,
1996
Total RNA aliquots (7.5 µg) were run on 1.4% (w/v) agarose denaturing
gel as described by Lehrach et al.
(1977 Specificity of the Trx h probes was ascertained by a dot-blot analysis. One nanogram of each plasmid containing a pea Trx h sequence was denaturated by 0.4 M NaOH, dotted in the edge of each blot, and UV cross linked.
The coding sequences of pea Trx h3 and h4 were subcloned in the BsaI site of the pASK-IBA3 plasmid (IBA, Gottingen, Germany) using the primers h3pASK-sens/h3pASK-anti and h4pASK-sens/h4pASK-anti, respectively (Table I), which were designed with the Primer D'Signer software (IBA, Gottingen, Germany). The pASK-IBA3 plasmid was designed to express target proteins fused to a C-terminal tag, allowing the purification of recombinant on Strep-Tactin resin (IBA).
The recombinant plasmids were introduced into competent E. coli
XL2 blue (Hanahan, 1983
For soluble protein extraction, embryo axes and cotyledons from pea seeds
imbibed for 0 to 46 h were ground in liquid nitrogen and 5 mL g fresh
weight–1 (or 10 mL for tissues from dry seeds) of
50 mM Tris-HCl (pH 7.8), 5 mM EDTA, and 5 mM
2-mercaptoethanol was added. The resulting homogenates were centrifuged
(50,000g for 20 min at 4°C), and the soluble proteins were stored
at –20°C. Protein contents were determined by using bovine serum
albumin as a standard (Bradford,
1976
For western-blot analysis, 15 to 50 µg of proteins was resolved per lane
on 12% or 15% (w/v) acrylamide gels by SDS-PAGE
(Laemmli, 1970 Rabbit antibodies raised against pea Trx h3 and h4 were produced by Davids Biotechnologie (Regensburg, Germany). Rabbit antibodies raised against Arabidopsis NTR were a gift of Y. Meyer (Unité Mixte de Recherche 5096, Centre National de la Recherche Scientifique, University of Perpignan, France).
Freshly separated embryo axes and cotyledons from pea seeds imbibed for 0
to 46 h were ground at room temperature in 0.1 M NaOH for the
extraction of NADPH (16 mL g–1 dry seeds or 12 mL
g–1 imbibed seeds) according to Zhao et al.
(1987
We thank Dr Julia Buitink for stimulating discussions and suggestions with the manuscript. Received December 21, 2002; returned for revision January 15, 2003; accepted March 14, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.102.019562.
1 This work was supported by the Conseil Régional Pays de Ra Loire,
France (CER Semences 2000–2003). * Corresponding author; e-mail francoise.montrichard{at}univangers.fr; fax 33–2–41739309.
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TOP
ABSTRACT
RESULTS
-amylase, pullulanase, and proteases and
the reduction of storage proteins, resulting in the degradation of
carbohydrate and protein reserves (Kobrehel et al.,
1991
) and water content (
) of seeds, allowed to germinate
on wet filter paper at 20°C in the dark, are indicated for different times
after the beginning of imbibition.
