sion of senescence from lower to upper leaves, and manyfactors involved in senescence have already been docu-mented. Furthermore, the loss of chlorophyll (Chl) is anearly, 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 putativeprotein product has a high homology to the early light-induced protein (ELIP), is up-regulated during leaf senescence. This mRNA (called ELIP-TOB) appearsearlier after addition of 1-amino-cyclopropane-1-carboxylic acid (ACC), which stimulates senescence,and is prevented by benzyladenine (BA), which delayssenescence. Furthermore, the appearance of ELIP-TOBmRNA is dependent on light, and is accelerated bystresses such as anaerobiosis and drought even beforeChl losses are detected. Thus, we show for the ®rst timethat ELIP is not only produced during greening in theearly stages of leaf development, but also duringsenescence. We present the possibility that elevatedlevels of ELIP-TOB mRNA at this later stage may be aresult, 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®cialsenescence,fullyexpandedgreenleaveswereremovedfromplantsthatwereapproximately8weeksoldandthebaseoftheleavesinsertedintodistilledwateroraqueoussolutionsofACC(1 mM)orBA (0.1 mM). Anaerobic conditions were obtained by submergingdetached leaves from 8-week-old plants under water. Water stresswas accomplished by withholding water from 8-week-old plants for10 d before leaf removal. The progression of senescence wasdetermined by comparing Chl content of the experimental materialto 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 untiluse. Total RNA was extracted according to the method of Puissantand Houdebine (1990). The concentration of RNA was determinedspectrophotometrically and resolved on agarose gels to evaluatequality. Polyadenylated RNA was prepared using the PolyATractRNA Isolation System (Promega). Double-stranded DNA wassynthesized with a cDNA Synthesis Module (Amersham) with ananchored oligo dT
25
that enabled the production of a cDNA librarycontaining a high percentage of full-length cDNA clones. Theyields of cDNA were estimated by the incorporation of [
32
P]dATP.
Treatment of cDNA for AFLP analysis
Samples (100 ng) of double-stranded cDNA were digested withEco
RI
and Mse
I
restriction enzymes (2.5 U each) and ligated toEco
RI
/Mse
I
adapters as described in the AFLP Analysis System Ikit protocol (Gibco BRL). The ligated fragments were preampli®edby including a single selective nucleotide at the 3
¢
end (Eco
RI
andMse
I
+ N) (where N represents A, C, G or T). This was followedby selective AFLP ampli®cation using Eco
RI
and Mse
I
+ NNNprimers labeled by phosphorylating the 5
¢
end with [
c
-
33
P]ATP.Polymerase chain reaction was performed as follows: the ®rst cyclewas 30 s at 94
°
C, 30 s at65
°
Cand1 minat72
°
C.Inthefollowing12 cycles the annealing temperature was reduced by 0.7
°
C for eachcycle. The ®nal 23 cycles were performed using the followingtemperatures: 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 anequal volume of 98% (v/v) formamide, 10 mM EDTA, 0.25%bromophenol blue, 0.25% xylene cyanol, heated for 3 min at 90
°
Cand immediately cooled on ice. A 2-
l
l portion of each sample wasresolved on a 6% (w/v) polyacrylamide sequencing gel. The PCRreaction products, using the same selective primers from young andsenescing leaves of tobacco, were resolved side by side. The gel wasthen dried and exposed to New Kodak BioMax MR ®lm overnightat room temperature.
Cloning of ampli®ed fragments
Senescence-associated bands were cut from the dried gel, soaked in50
l
l sterile water and heated for 2 h at 65
°
C. After a brief centrifugation, 10
l
l of the supernatant was transferred to anothertube. Re-ampli®cation of the recovered fragment was performedwiththesameprimersinitiallyusedfortheselectiveAFLPreactions.The temperature pro®le for PCR (30 cycles) was as follows: 30 s at94
°
C, 30 s at 56
°
C, and 1 min at 72
°
C. The PCR products wereseparated on a 2% (w/v) agarose gel, then eluted and ligated topUC57 vector in order to transform competent DH5 alpha cells. Toverify 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 leaveswas 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
3 M NaCl, 0.3 M sodiumcitrate, pH 8.0) as instructed. For preparation of probes, plasmid-transformed cells containing the DNA fragment of interest werecultured overnight in 5 ml LB (Sambrook et al. 1989) thatcontained 100
l
g/ml ampicillin at 37
°
C. After plasmid isolation,the fragment was then ampli®ed under the same conditions as thosedescribed for the ampli®cation of DNA fragments from dried gels.The fragment was eluted from the agarose gel and labeled by therandom primer labeling technique using [
32
P]dATP as described inSambrooketal.(1989). HybridizationofRNA blotswas performedovernight in a mixture of 6
´
SSC, 0.5% (w/v) SDS, 50% (v/v)formamide, 0.1 mg/ml of sonicated and denatured salmon spermDNA, and 2
´
10
6
cpm/ml of probe at 60
°
C. After hybridization,the blot was washed successively with 2
´
SSC, 0.1% (w/v) SDS for15 min at 55
°
C, and1
´
SSC, 0.1%(w/v) SDS for15 min at55
°
C,and 0.5
´
SSC, 0.1%(w/v) SDS for 15 min at 55
°
C.
Immunoblots
Fifty micrograms of total leaf protein in buer containing SDS wasseparated by PAGE and blotted as described previously (Dumbro and Gepstein 1993). The blots were exposed to antibodies elicitedagainst tobacco ELIP (produced and provided by Prof. K.Kloppstech), washed, and then treated with goat anti-rabbit IgGconjugated to peroxidase (BioRad). Binding was visualized bytreatment with peroxidase substrate.592 L. Binyamin et al.: Production of early light-induced protein during senescence
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