Planta (2001) 212: 591±597
The early light-induced protein is also produced during leaf
senescence of Nicotiana tabacum
L. Binyamin, M. Falah, V. Portnoy, E. Soudry, S. Gepstein
Faculty of Biology, Technion, Israel Institute of Technology, Haifa, 32000, Israel
Received: 18 May 2000 / Accepted: 24 June 2000
Abstract. To better understand the genetic controls of
leaf senescence, a tobacco (Nicotiana tabacum L. cv.
SR1) mRNA that is up-regulated during senescence
was isolated by the cDNA-ampli®ed restriction frag-
ment polymorphism method and the cDNA was
cloned. The mRNA coded for the early light-induced
protein (ELIP), a member of the chlorophyll a/b-
binding protein family that has been implicated in
assembly or repair of the photosynthetic machinery
during early chloroplast development and abiotic stress.
A protein antigenically recognized by antibodies to
ELIP appeared during senescence with kinetics similar
to those of its mRNA. The mRNA, designated ELIP-
TOB, was detected earlier when senescence was en-
hanced by leaf detachment and treatment with 1-ami-
no-cyclopropane-1-carboxylic acid, and was detected
later when senescence was retarded by benzyladenine.
However, no ELIP-TOB mRNA was seen in the dark
even though senescence was accelerated under these
conditions. Furthermore, water stress and anaerobiosis
stimulated the appearance of ELIP-TOB mRNA before
losses of chlorophyll could be detected. We discuss the
conditions that may lead to the up-regulation of ELIP-
TOB during senescence and speculate as to the role of
the gene product in this terminal phase of leaf
development.
Key words: Abiotic stress ± Chloroplast ± Early light-
induced protein ± Nicotiana (leaf senescence) ±
Senescence
Abbreviations: ACC ˆ 1-amino-cyclopropane-1-carboxylic acid;
BA ˆ benzyladenine; cDNA-AFLP ˆ ampli®ed restriction frag-
ment polymorphism-derived technique for RNA ®nger-printing;
Chl ˆ chlorophyll; ELIP ˆ early light-induced protein; PCR ˆ
polymerase chain reaction
Correspondence to: S. Gepstein;
E-mail: gepstein@tx.technion.ac.il; Fax:+972-4-8225135
Introduction
Leaf senescence, the ®nal stage of leaf development, is
controlled in part by a program involving gene expres-
sion (Becker and Apel 1993; Buchanan-Wollaston 1997;
Smart 1994; Humbeck et al. 1996; Gan and Amasino
1997). The ensuing synthesis of particular proteins is
responsible for the degradation of essential macromol-
ecules, profound alterations of cell structure and phys-
iology, the mobilization of hydrolytic products to other
parts of the plant, and the eventual death of the organ
(Thimann 1980; Nooden 1988; Guarente et al. 1998;
Weaver et al. 1998). The chloroplast appears to be an
early target for senescence processes (Gepstein 1988).
In order to understand the complex operations
involved in cell death, it is essential to identify the
important components of its genetic program, even those
that may not be strictly causal for senescence. One
approach is to isolate mRNAs that are up-regulated
during senescence, and then predict their function by
homology to known gene products. The relationship of
these proteins to senescence eventually can be determined
by other molecular, cellular and biochemical techniques.
With this objective in mind, di€erential screening and
di€erential display techniques have resulted in the
identi®cation of a variety of senescence-associated genes
that provide greater insight into the processes related to
cell death (Smart 1994; Buchanan-Wollaston 1997; Gan
and Amasino 1997). This in turn led to the suggestion
that the regulation of senescence involves several inter-
connected genetic pathways (Gan and Amasino 1997).
We have been identifying other senescence-associated
genes using an RNA ®ngerprinting technique based on
the method of ampli®ed restriction fragment polymor-
phism (cDNA-AFLP) (Bachem et al. 1996). The advan-
tage of this technique is that oligonucleotide adapters
can be used as primer sites for the polymerase chain
reaction (PCR). As a result, the PCR conditions are
more stringent, and PCR artifacts that are common in
di€erential display are decreased (Bachem et al. 1996).
For this study, tobacco leaves were chosen as the
experimental material. They show a predictable progres-
592 L. Binyamin et al.: Production of early light-induced protein during senescence
sion of senescence from lower to upper leaves, and many
factors involved in senescence have already been docu-
mented. Furthermore, the loss of chlorophyll (Chl) is an
early, easily measured, and accepted criterion for senes-
cence in tobacco (Aharoni and Liberman 1979). Finally,
this plant is amenable to a range of molecular tech-
niques, including the production of transgenic plants.
We show here that a tobacco mRNA, whose putative
protein product has a high homology to the early light-
induced protein (ELIP), is up-regulated during leaf
senescence. This mRNA (called ELIP-TOB) appears
earlier after addition of 1-amino-cyclopropane-1-
carboxylic acid (ACC), which stimulates senescence,
and is prevented by benzyladenine (BA), which delays
senescence. Furthermore, the appearance of ELIP-TOB
mRNA is dependent on light, and is accelerated by
stresses such as anaerobiosis and drought even before
Chl losses are detected. Thus, we show for the ®rst time
that ELIP is not only produced during greening in the
early stages of leaf development, but also during
senescence. We present the possibility that elevated
levels of ELIP-TOB mRNA at this later stage may be a
result, at least in part, of stress perception by leaf cells.
Materials and methods
Plant material
Tobacco (Nicotiana tabacum L. cv. SR1) plants were grown in soil-
containing pots in the greenhouse at an average temperature of
25 °C under a 16-h photoperiod maintained with ¯uorescent lights.
The plants were watered with tap water every 2 d. For studies of
arti®cial senescence, fully expanded green leaves were removed from
plants that were approximately 8 weeks old and the base of the leaves
inserted into distilled water or aqueous solutions of ACC (1 mM) or
BA (0.1 mM). Anaerobic conditions were obtained by submerging
detached leaves from 8-week-old plants under water. Water stress
was accomplished by withholding water from 8-week-old plants for
10 d before leaf removal. The progression of senescence was
determined by comparing Chl content of the experimental material
to that of green, fully expanded leaves using the method of Moran
(1982). The various stages of senescence were designated as: G2,
100% (Chl content); G1, 80%; GY, 60%, Y1, 50%; Y2, 25%.
Preparation of poly(A)+RNA and the synthesis of cDNA
Leaves were frozen in liquid nitrogen and stored at )80 °C until
use. Total RNA was extracted according to the method of Puissant
and Houdebine (1990). The concentration of RNA was determined
spectrophotometrically and resolved on agarose gels to evaluate
quality. Polyadenylated RNA was prepared using the PolyATract
RNA Isolation System (Promega). Double-stranded DNA was
synthesized with a cDNA Synthesis Module (Amersham) with an
anchored oligo dT25 that enabled the production of a cDNA library
containing a high percentage of full-length cDNA clones. The
yields of cDNA were estimated by the incorporation of [32P]dATP.
Treatment of cDNA for AFLP analysis
Samples (100 ng) of double-stranded cDNA were digested with
Eco RI and Mse I restriction enzymes (2.5 U each) and ligated to
Eco RI/Mse I adapters as described in the AFLP Analysis System I
kit protocol (Gibco BRL). The ligated fragments were preampli®ed
by including a single selective nucleotide at the 3¢ end (Eco RI and
Mse I + N) (where N represents A, C, G or T). This was followed
by selective AFLP ampli®cation using Eco RI and Mse I + NNN
primers labeled by phosphorylating the 5¢ end with [c-33P]ATP.
Polymerase chain reaction was performed as follows: the ®rst cycle
was 30 s at 94 °C, 30 s at 65 °C and 1 min at 72 °C. In the following
12 cycles the annealing temperature was reduced by 0.7 °C for each
cycle. The ®nal 23 cycles were performed using the following
temperatures: 30 s at 94 °C, 30 s at 56 °C, and 1 min at 72 °C.
Gel electrophoresis
The reaction mixtures of the PCR products were combined with an
equal volume of 98% (v/v) formamide, 10 mM EDTA, 0.25%
bromophenol blue, 0.25% xylene cyanol, heated for 3 min at 90 °C
and immediately cooled on ice. A 2-ll portion of each sample was
resolved on a 6% (w/v) polyacrylamide sequencing gel. The PCR
reaction products, using the same selective primers from young and
senescing leaves of tobacco, were resolved side by side. The gel was
then dried and exposed to New Kodak BioMax MR ®lm overnight
at room temperature.
Cloning of ampli®ed fragments
Senescence-associated bands were cut from the dried gel, soaked in
50 ll sterile water and heated for 2 h at 65 °C. After a brief
centrifugation, 10 ll of the supernatant was transferred to another
tube. Re-ampli®cation of the recovered fragment was performed
with the same primers initially used for the selective AFLP reactions.
The temperature pro®le for PCR (30 cycles) was as follows: 30 s at
94 °C, 30 s at 56 °C, and 1 min at 72 °C. The PCR products were
separated on a 2% (w/v) agarose gel, then eluted and ligated to
pUC57 vector in order to transform competent DH5 alpha cells. To
verify that the isolated fragment was indeed senescence-associated,
it was utilized as a probe for Northern blot analysis.
Hybridization of RNA blots
Fifteen micrograms of total RNA from young and senescing leaves
was denatured with glyoxal, subjected to electrophoresis on a 1%
(w/v) agarose gel, and transferred to a Zeta-Probe GT membrane
(Bio-Rad) in 20 ´ SSC (20 ´ SSC ˆ 3 M NaCl, 0.3 M sodium
citrate, pH 8.0) as instructed. For preparation of probes, plasmid-
transformed cells containing the DNA fragment of interest were
cultured overnight in 5 ml LB (Sambrook et al. 1989) that
contained 100 lg/ml ampicillin at 37 °C. After plasmid isolation,
the fragment was then ampli®ed under the same conditions as those
described for the ampli®cation of DNA fragments from dried gels.
The fragment was eluted from the agarose gel and labeled by the
random primer labeling technique using [32P]dATP as described in
Sambrook et al. (1989). Hybridization of RNA blots was performed
overnight in a mixture of 6 ´ SSC, 0.5% (w/v) SDS, 50% (v/v)
formamide, 0.1 mg/ml of sonicated and denatured salmon sperm
DNA, and 2 ´ 106 cpm/ml of probe at 60 °C. After hybridization,
the blot was washed successively with 2 ´ SSC, 0.1% (w/v) SDS for
15 min at 55 °C, and 1 ´ SSC, 0.1% (w/v) SDS for 15 min at 55 °C,
and 0.5 ´ SSC, 0.1%(w/v) SDS for 15 min at 55 °C.
Immunoblots
Fifty micrograms of total leaf protein in bu€er containing SDS was
separated by PAGE and blotted as described previously (Dumbro€
and Gepstein 1993). The blots were exposed to antibodies elicited
against tobacco ELIP (produced and provided by Prof. K.
Kloppstech), washed, and then treated with goat anti-rabbit IgG
conjugated to peroxidase (BioRad). Binding was visualized by
treatment with peroxidase substrate.
L. Binyamin et al.: Production of early light-induced protein during senescence
Results
Using the cDNA-AFLP technique, we isolated and
cloned a 247-bp cDNA that binds to an mRNA of
approximately 800 bp and that increases during senes-
cence. This cDNA is a fragment with a high similarity
(79%) to the ELIP family of proteins, nuclear-encoded
chloroplast proteins, whose appearance is dependent on
the presence of light (Meyer and Kloppstech 1984;
Grimm and Kloppstech 1987). The region of the
deduced ELIP amino acid sequence from tobacco is
homologous to the most conserved region of ELIPs
from pea, soybean and Arabidopsis (Fig. 1). Because the
amino acid sequence has a high homology to ELIPs, we
called the isolated fragment ELIP-TOB.
As shown in Fig. 2A, the ELIP-TOB cDNA bound
to an mRNA that appeared when Chl levels had
decreased by about 40% (GY). The mRNA levels
increased until Chl levels were 50% or less than the
original value (Y1). A northern blot was also performed
using RNA from detached leaves that began to senesce
within 1 to 3 d (Fig. 2B). The trend is similar to that of
the intact leaves that age more slowly, but the ELIP-
TOB mRNA appeared only 1 d after detachment, even
before Chl levels began to decline. In fact, it was possible
to detect an increase in the mRNA only 4 h after leaf
removal (see Fig. 4). As Chl content decreased, there
was a striking enhancement of this mRNA (Fig. 2B).
To determine if the ELIP protein was produced
during leaf senescence, antibodies were obtained in
order to run immunoblots. The results (Fig. 2C) indicate
that ELIP protein is detectable in the tobacco leaves
after Chl levels have decreased about 40%.
To test whether the appearance of ELIP-TOB mRNA
during senescence is dependent on light, detached tobacco
leaves were aged in the dark, and the steady-state levels of
this mRNA were estimated by Northern blots (Fig. 3A).
There was no detectable mRNA from the ELIP-TOB
gene in leaves incubated in the dark compared with those
in the light, even though the dark treatment resulted in a
more rapid loss of chlorophyll. This experiment strongly
suggests that the increased levels of ELIP-TOB mRNA
during senescence are dependent on light.
To further examine the light requirement for the
senescence-induced up-regulation of ELIP-TOB, intact
Fig. 1. Alignment of ELIP amino acid sequences. Amino acid
sequences deduced from the corresponding partial cDNA sequence
of ELIP-TOB are compared with the ®rst transmembrane domain of
ELIP proteins from soybean (Soy) (accession number JC 5876), pea
(Pea) (accession numbers P11432, SO1056), and Arabidopsis (Arb)
(accession number 88391). Also shown is the sequence of Sep1 from
593
tobacco plants were transferred to darkness for 20 d, and
the ELIP-TOB mRNA levels of senescing leaves from
these plants were compared with senescing leaves from
similarly aged plants that remained in the light (Fig. 3B).
After 20 d in the dark, the mRNA for ELIP-TOB could
not be detected in the senescent leaves, but it was seen in
senescent leaves of plants in the light even though the
light-treated leaves chosen showed the same degree of
Chl loss as those in the dark. In another experiment,
detached leaves were incubated in the light until the GY
stage and then placed in the dark for 2 d longer (Fig. 3C).
Although ELIP-TOB mRNA was present at the GY
stage (Fig. 2B), the mRNA was undetectable after dark
incubation even though only 50% of the Chl remained
(stage Y1); ELIP-TOB mRNA was clearly present at this
same stage in light-treated leaves (Fig. 3C).
We also studied the e€ect of certain hormones that
regulate senescence on the levels of ELIP-TOB mRNA
in green, detached leaves. A northern blot (Fig. 4)
showed that treatment with ACC, the precursor of
ethylene, clearly increased the mRNA after only 2 h,
and the level remained high for 10 d. On the other hand,
the synthetic cytokinin, BA, delayed the increase in
ELIP-TOB mRNA until about day 3, and only at day 5
did the level of this mRNA approach that of the
control.
Expression of ELIPs has been associated with certain
stresses (Adamska et al. 1992; Potter and Kloppstech
1993; Adamska 1997). To compare mRNA levels of
ELIP-TOB with those of other plants under stress, we
exposed detached tobacco leaves to anaerobic condi-
tions (Fig. 5A). Chlorophyll levels decreased at a slower
rate than leaves in air, as seen previously for pathogen-
induced programmed cell death (Mittler et al. 1996), but
steady-state levels of ELIP-TOB mRNA increased
markedly by 1 d after the oxygen stress and remained
elevated. Levels of ELIP-TOB mRNA in controls
reached a maximum only at day 5 (Figs. 2B, 4).
The plants were also subjected to a drought stress
(Fig. 5B). There was a marked increase in ELIP-TOB
mRNA in leaves of plants without water for 10 d,
whereas this mRNA was undetectable in control leaves
of the same age from plants that had sucient water.
The water stress resulted in an elevation of ELIP-TOB
mRNA levels before Chl decreases could be measured.
Arabidopsis (Arb-Sep1) (accession number AAF61625). Dashes
indicate the gaps inserted to allow optimal alignment. The black
boxes indicate identical residues and gray boxes indicate conservative
substitutions Alignment of ELIPs and Sep was carried out using the
multiple alignment program, CLUSTLAW
594 L. Binyamin et al.: Production of early light-induced protein during senescence
Fig. 2 A±C. Steady-state levels of ELIP-TOB mRNA and ELIP
protein during senescence of tobacco leaves. A Northern blot of total
RNA, using leaves from a mature tobacco plant. Greener leaves (G2,
G1) were removed from the top of the plant; more-yellow leaves (YG,
Y1) are from the bottom. B Northern blot of total RNA from
detached leaves incubated in the light on water-soaked ®lter paper.
Time after detachment is indicated at the top of each lane; stages of
senescence, as determined by Chl levels, are indicated at the bottom of
Fig. 3A±C. E€ects of light and darkness on the appearance of ELIP-
TOB mRNA. A Northern blot of total RNA extracted from detached
leaves at times indicated at the top of each lane. Leaves were
incubated in continuous darkness (D) or in continuous light (L).
B Northern blot of total RNA extracted from mature, fully expanded
tobacco leaves (0) or from leaves at the same position after a further
Discussion
Using the cDNA-AFLP technique to search for mRNAs
that are up-regulated during senescence, we found a
particular mRNA whose steady-state level increased as
each lane. C Western blot, using polyclonal ELIP antibodies. Total
protein was extracted from detached leaves at the indicated stages of
senescence. Location and intensity of the antigen are indicated by the
peroxidase reaction. Stages of senescence, as determined by chloro-
phyll levels, are indicated at the bottom of each lane. Stages: G2,
100% of the chlorophyll of a fully expanded green leaf; G1, 80%; GY,
60%; Y1, 50%; Y2, 25%. Total RNA, as indicated by methylene blue
staining, is shown below the lines in A and B
20 d in continuous darkness (D) or continuous light (L). C Northern
blot of total RNA extracted from detached leaves (initially at GY
stage) that had been incubated for a further 2 d in the dark (D) or
light (L). In all panels, total RNA, as indicated by methylene blue
staining, is shown below the line. Stages of senescence are indicated at
the bottom of each lane (see Fig. 2 for an explanation)
Chl decreased in tobacco leaves (Fig. 2). The cDNA-
AFLP-derived probe used to identify the mRNA is a
fragment whose putative translation product has a high
homology to ELIPs (Fig. 1), members of a family of
light-dependent, stress-induced Chl-binding proteins
L. Binyamin et al.: Production of early light-induced protein during senescence
Fig. 4. E€ect of senescence-modifying agents on the appearance of
ELIP-TOB mRNA. Detached tobacco leaves were incubated in
continuous light on ®lter paper soaked in distilled water, 1 mM ACC
or 0.1 mM BA. Time after detachment is indicated on the left.
Methylene blue staining for total RNA (approximately equal for all
treatments) has been omitted in order to simplify the ®gure
Fig. 5A,B. E€ect of anaerobiosis and drought on the appearance of
ELIP-TOB mRNA in tobacco leaves. A Northern blot using total
RNA from detached leaves submerged in distilled water and incubated
in continuous light. The RNA was extracted at times indicated above
each lane. Stages of senescence are indicated at the bottom of each
lane. B Levels of ELIP-TOB mRNA from mature green leaves (G2) of
plants before (0) or 10 d after water deprivation (10d) (two lanes on the
595
(Kolanus et al. 1987). The ELIP-TOB aligns well with
segment I of the three potential transmembrane seg-
ments of the full-length ELIP protein (Fig. 1) (Grimm
et al. 1989; Green and Kuhlbrandt 1995). Furthermore,
the approximately 800-bp size of the RNA (data not
shown) corresponds to the 24- to 25-kDa size of the
putative translated ELIP protein (Meyer and Klopps-
tech 1984; Scharnhorst et al. 1985). The light require-
ment for ELIP-TOB gene expression (Fig. 3) implies
that a similarity also exists in the regulatory region of
the gene (Blecken et al. 1994). The probe for ELIP-TOB
presumably would not hybridize with the mRNA for
Seps (Fig. 1), stress-related members of the ELIP group
from Arabidopsis described by Heddad and Adamska
(2000), since Seps have only two transmembrane do-
mains and are induced by light stress with di€erent
kinetics as compared with ELIPs.
Up to now, ELIPs were reported to be associated
mainly with chloroplast biogenesis during leaf greening
(Meyer and Kloppstech 1984; Grimm et al. 1989) and
during environmental stresses, such as high light (Klopps-
tech et al. 1991; Adamska et al. 1992, 1993), UV-A
(Adamska et al. 1992), temperature (Adamska and
Kloppstech 1994), and drought (Ouvrard et al. 1996).
Humbeck et al. (1996) reported that under light stress,
ELIP expression is independent of the developmental
stages of barley ¯ag leaves, and that after the onset of
senescence ELIP mRNA and protein still accumulate. We
report here that both the mRNA for this protein and a
protein that is recognized by ELIP antibodies are up-
regulated during the natural senescence of attached leaves
(Fig. 2) even under low light intensity. Furthermore,
enhancement of senescence by detaching the leaf
(Thimann 1980; Fig. 2) or by addition of ACC (Gepstein
and Thimann 1981; Grbic and Bleecker 1995) leads to an
left). The ELIP-TOB mRNA from mature green leaves (G2) removed
from plants watered normally during the 10 d period (10d) is shown in
the third lane (to the right of the dotted line). The RNA from a
yellowing leaf (Y1) from a watered plant (fourth lane from the left) is
used to show normal induction during senescence. In both panels, total
RNA, as indicated by methylene blue staining, is shown below the line.
See Fig. 2 for an explanation of the senescence stages
596 L. Binyamin et al.: Production of early light-induced protein during senescence
up-regulation of the ELIP-TOB mRNA, whereas retar-
dation of senescence by BA (van Staden et al. 1988; Smart
1994) delays this up-regulation (Fig. 4).
It appears, however, that ELIP is not essential for the
Chl loss that is characteristic of the senescence syn-
drome. This conclusion is based on the absence of ELIP-
TOB mRNA in the dark in attached or detached leaves,
even though senescence is accelerated under these
conditions (Fig. 3). What is more, this mRNA is up-
regulated by anaerobiosis and water stress even before
decreases in Chl can be detected (Fig. 5). It is possible
that the appearance of ELIP-TOB mRNA in isolated
leaves before the loss of Chl is due to a mechanical stress
associated with detachment. Interestingly, mRNAs of
some other members of the chlorophyll a/b-binding
protein (CAB) family have kinetics opposite to those of
ELIP (Adamska and Kloppstech 1991; Bei-Paraskevo-
poulou and Kloppstech 1999). Expression of the gene
for light-harvesting chlorophyll (LHC), for example, is
very low in developing leaves, reaches a maximum in
mature leaves, and then decreases during senescence
(Roberts et al. 1987; Gepstein 1988; Bate et al. 1990).
The appearance of ELIP-TOB mRNA and protein
during senescence adds the ELIP gene to the list of
senecsence-associated genes, but, as noted above, there is
a lack of correlation with Chl loss. Perhaps ELIP-TOB is
responding to the same signals occurring during senes-
cence that also occur during chloroplast development or
stress, e.g. modi®cations of pigment/membrane associ-
ations a€ecting the degree of reduction of electron-chain
components (Montane et al. 1998). This implies that the
appearance of ELIP is due to a senescence-associated
stress perceived by leaf cells. Another example that may
parallel the behavior of ELIP during senescence is the
light-dependent induction of ELIP upon desiccation of
Craterostigma plantaginum (Bartels et al. 1992).
The function of ELIP during senescence remains
unclear. Like other CAB family members, ELIP is
localized to the chloroplast, but unlike other family
members that are membrane-bound components of
photosystems I and II, the ELIP protein is present only
transiently in the thylakoid membranes (Grimm et al.
1989; Heddad and Adamska 2000). However, ELIP has
been shown to bind Chl and lutein, thereby implying
that ELIP may be a pigment scavenger (Adamska et al.
1999). As a result, the Chl may be destroyed by speci®c
enzymes or inserted into the thylakoid membrane
(Adamska 1997). A protein with these properties might
be expected to play a role during early leaf development
or during various abiotic stresses when there is an
increased pigment-protein turnover. These are in fact the
times at which ELIP is most abundant (Adamska 1997).
The hypothesized functions of ELIP as a pigment-
carrier may be applied to senescence. Perhaps during
senescence the newly synthesized ELIP binds free Chl
released from LHCII in the same manner as during
stress conditions, which lead to a degradation of the
pigment molecules. The transient expression of ELIP
during greening and the characteristic decrease in
LHCII proteins as ELIP increases during senescence
are consistent with this speculation.
Alternatively, ELIP may help to maintain chloroplast
function during senescence by a process similar to that
occurring at chloroplast development ± the addition of
Chl to proteins in the membrane (Adamska 1997).
However, other simultaneously occurring degradative
processes eventually overcome this function. As a result,
one might speculate further that the presence of ELIP
only in the light may help account for the well-known
retardation of senescence by illumination of detached
leaves.
We are grateful to Prof. Klaus Kloppstech (University of
Hannover) for providing tobacco ELIP antibodies. Thanks also
to Prof. Bernard Rubinstein for discussions during the preparation
of this manuscript.
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