sion of senescence from lower to upper leaves, and many by including a single selective nucleotide at the 3¢ end (Eco RI and
factors involved in senescence have already been docu- 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
mented. Furthermore, the loss of chlorophyll (Chl) is an
primers labeled by phosphorylating the 5¢ end with [c-33P]ATP.
early, easily measured, and accepted criterion for senes- Polymerase chain reaction was performed as follows: the ®rst cycle
cence in tobacco (Aharoni and Liberman 1979). Finally, was 30 s at 94 °C, 30 s at 65 °C and 1 min at 72 °C. In the following
this plant is amenable to a range of molecular tech- 12 cycles the annealing temperature was reduced by 0.7 °C for each
niques, including the production of transgenic plants. cycle. The ®nal 23 cycles were performed using the following
We show here that a tobacco mRNA, whose putative temperatures: 30 s at 94 °C, 30 s at 56 °C, and 1 min at 72 °C.
protein product has a high homology to the early light-
induced protein (ELIP), is up-regulated during leaf Gel electrophoresis
senescence. This mRNA (called ELIP-TOB) appears
earlier after addition of 1-amino-cyclopropane-1- The reaction mixtures of the PCR products were combined with an
carboxylic acid (ACC), which stimulates senescence, equal volume of 98% (v/v) formamide, 10 mM EDTA, 0.25%
and is prevented by benzyladenine (BA), which delays bromophenol blue, 0.25% xylene cyanol, heated for 3 min at 90 °C
senescence. Furthermore, the appearance of ELIP-TOB and immediately cooled on ice. A 2-ll portion of each sample was
resolved on a 6% (w/v) polyacrylamide sequencing gel. The PCR
mRNA is dependent on light, and is accelerated by reaction products, using the same selective primers from young and
stresses such as anaerobiosis and drought even before senescing leaves of tobacco, were resolved side by side. The gel was
Chl losses are detected. Thus, we show for the ®rst time then dried and exposed to New Kodak BioMax MR ®lm overnight
that ELIP is not only produced during greening in the at room temperature.
early stages of leaf development, but also during
senescence. We present the possibility that elevated Cloning of ampli®ed fragments
levels of ELIP-TOB mRNA at this later stage may be a
result, at least in part, of stress perception by leaf cells. 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
Materials and methods tube. Re-ampli®cation of the recovered fragment was performed
with the same primers initially used for the selective AFLP reactions.
Plant material 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
Tobacco (Nicotiana tabacum L. cv. SR1) plants were grown in soil- separated on a 2% (w/v) agarose gel, then eluted and ligated to
containing pots in the greenhouse at an average temperature of pUC57 vector in order to transform competent DH5 alpha cells. To
25 °C under a 16-h photoperiod maintained with ¯uorescent lights. verify that the isolated fragment was indeed senescence-associated,
The plants were watered with tap water every 2 d. For studies of it was utilized as a probe for Northern blot analysis.
arti®cial senescence, fully expanded green leaves were removed from
plants that were approximately 8 weeks old and the base of the leaves
Hybridization of RNA blots
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 Fifteen micrograms of total RNA from young and senescing leaves
was accomplished by withholding water from 8-week-old plants for was denatured with glyoxal, subjected to electrophoresis on a 1%
10 d before leaf removal. The progression of senescence was (w/v) agarose gel, and transferred to a Zeta-Probe GT membrane
determined by comparing Chl content of the experimental material (Bio-Rad) in 20 ´ SSC (20 ´ SSC 3 M NaCl, 0.3 M sodium
to that of green, fully expanded leaves using the method of Moran citrate, pH 8.0) as instructed. For preparation of probes, plasmid-
(1982). The various stages of senescence were designated as: G2, transformed cells containing the DNA fragment of interest were
100% (Chl content); G1, 80%; GY, 60%, Y1, 50%; Y2, 25%. 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.
Preparation of poly(A)+RNA and the synthesis of cDNA
The fragment was eluted from the agarose gel and labeled by the
random primer labeling technique using [32P]dATP as described in
Leaves were frozen in liquid nitrogen and stored at )80 °C until Sambrook et al. (1989). Hybridization of RNA blots was performed
use. Total RNA was extracted according to the method of Puissant overnight in a mixture of 6 ´ SSC, 0.5% (w/v) SDS, 50% (v/v)
and Houdebine (1990). The concentration of RNA was determined formamide, 0.1 mg/ml of sonicated and denatured salmon sperm
spectrophotometrically and resolved on agarose gels to evaluate DNA, and 2 ´ 106 cpm/ml of probe at 60 °C. After hybridization,
quality. Polyadenylated RNA was prepared using the PolyATract the blot was washed successively with 2 ´ SSC, 0.1% (w/v) SDS for
RNA Isolation System (Promega). Double-stranded DNA was 15 min at 55 °C, and 1 ´ SSC, 0.1% (w/v) SDS for 15 min at 55 °C,
synthesized with a cDNA Synthesis Module (Amersham) with an and 0.5 ´ SSC, 0.1%(w/v) SDS for 15 min at 55 °C.
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. Immunoblots
Fig. 1. Alignment of ELIP amino acid sequences. Amino acid Arabidopsis (Arb-Sep1) (accession number AAF61625). Dashes
sequences deduced from the corresponding partial cDNA sequence indicate the gaps inserted to allow optimal alignment. The black
of ELIP-TOB are compared with the ®rst transmembrane domain of boxes indicate identical residues and gray boxes indicate conservative
ELIP proteins from soybean (Soy) (accession number JC 5876), pea substitutions Alignment of ELIPs and Sep was carried out using the
(Pea) (accession numbers P11432, SO1056), and Arabidopsis (Arb) multiple alignment program, CLUSTLAW
(accession number 88391). Also shown is the sequence of Sep1 from
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 each lane. C Western blot, using polyclonal ELIP antibodies. Total
protein during senescence of tobacco leaves. A Northern blot of total protein was extracted from detached leaves at the indicated stages of
RNA, using leaves from a mature tobacco plant. Greener leaves (G2, senescence. Location and intensity of the antigen are indicated by the
G1) were removed from the top of the plant; more-yellow leaves (YG, peroxidase reaction. Stages of senescence, as determined by chloro-
Y1) are from the bottom. B Northern blot of total RNA from phyll levels, are indicated at the bottom of each lane. Stages: G2,
detached leaves incubated in the light on water-soaked ®lter paper. 100% of the chlorophyll of a fully expanded green leaf; G1, 80%; GY,
Time after detachment is indicated at the top of each lane; stages of 60%; Y1, 50%; Y2, 25%. Total RNA, as indicated by methylene blue
senescence, as determined by Chl levels, are indicated at the bottom of staining, is shown below the lines in A and B
Fig. 3A±C. Eects of light and darkness on the appearance of ELIP- 20 d in continuous darkness (D) or continuous light (L). C Northern
TOB mRNA. A Northern blot of total RNA extracted from detached blot of total RNA extracted from detached leaves (initially at GY
leaves at times indicated at the top of each lane. Leaves were stage) that had been incubated for a further 2 d in the dark (D) or
incubated in continuous darkness (D) or in continuous light (L). light (L). In all panels, total RNA, as indicated by methylene blue
B Northern blot of total RNA extracted from mature, fully expanded staining, is shown below the line. Stages of senescence are indicated at
tobacco leaves (0) or from leaves at the same position after a further the bottom of each lane (see Fig. 2 for an explanation)
Fig. 5A,B. Eect of anaerobiosis and drought on the appearance of left). The ELIP-TOB mRNA from mature green leaves (G2) removed
ELIP-TOB mRNA in tobacco leaves. A Northern blot using total from plants watered normally during the 10 d period (10d) is shown in
RNA from detached leaves submerged in distilled water and incubated the third lane (to the right of the dotted line). The RNA from a
in continuous light. The RNA was extracted at times indicated above yellowing leaf (Y1) from a watered plant (fourth lane from the left) is
each lane. Stages of senescence are indicated at the bottom of each used to show normal induction during senescence. In both panels, total
lane. B Levels of ELIP-TOB mRNA from mature green leaves (G2) of RNA, as indicated by methylene blue staining, is shown below the line.
plants before (0) or 10 d after water deprivation (10d) (two lanes on the 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- Alternatively, ELIP may help to maintain chloroplast
dation of senescence by BA (van Staden et al. 1988; Smart function during senescence by a process similar to that
1994) delays this up-regulation (Fig. 4). occurring at chloroplast development ± the addition of
It appears, however, that ELIP is not essential for the Chl to proteins in the membrane (Adamska 1997).
Chl loss that is characteristic of the senescence syn- However, other simultaneously occurring degradative
drome. This conclusion is based on the absence of ELIP- processes eventually overcome this function. As a result,
TOB mRNA in the dark in attached or detached leaves, one might speculate further that the presence of ELIP
even though senescence is accelerated under these only in the light may help account for the well-known
conditions (Fig. 3). What is more, this mRNA is up- retardation of senescence by illumination of detached
regulated by anaerobiosis and water stress even before leaves.
decreases in Chl can be detected (Fig. 5). It is possible
that the appearance of ELIP-TOB mRNA in isolated We are grateful to Prof. Klaus Kloppstech (University of
leaves before the loss of Chl is due to a mechanical stress Hannover) for providing tobacco ELIP antibodies. Thanks also
associated with detachment. Interestingly, mRNAs of to Prof. Bernard Rubinstein for discussions during the preparation
some other members of the chlorophyll a/b-binding of this manuscript.
protein (CAB) family have kinetics opposite to those of
ELIP (Adamska and Kloppstech 1991; Bei-Paraskevo-
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