Elips Leaf Sene

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 fragment polymorphism method and the cDNA was cloned. The mRNA coded for the early light-induced protein (ELIP), a member of the chlorophyll a/bbinding 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 ELIPTOB, was detected earlier when senescence was enhanced by leaf detachment and treatment with 1-amino-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 ELIPTOB 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 lightinduced protein ± Nicotiana (leaf senescence) ± Senescence

Abbreviations: ACC ˆ 1-amino-cyclopropane-1-carboxylic acid; BA ˆ benzyladenine; cDNA-AFLP ˆ ampli®ed restriction fragment 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: [email protected]; Fax:+972-4-8225135

Introduction Leaf senescence, the ®nal stage of leaf development, is controlled in part by a program involving gene expression (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 macromolecules, profound alterations of cell structure and physiology, 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 interconnected 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 polymorphism (cDNA-AFLP) (Bachem et al. 1996). The advantage 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-

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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 documented. Furthermore, the loss of chlorophyll (Chl) is an early, easily measured, and accepted criterion for senescence in tobacco (Aharoni and Liberman 1979). Finally, this plant is amenable to a range of molecular techniques, including the production of transgenic plants. We show here that a tobacco mRNA, whose putative protein product has a high homology to the early lightinduced protein (ELIP), is up-regulated during leaf senescence. This mRNA (called ELIP-TOB) appears earlier after addition of 1-amino-cyclopropane-1carboxylic 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 soilcontaining 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, plasmidtransformed 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 senescence. 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 ELIPTOB 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

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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 conditions (Fig. 5A). Chlorophyll levels decreased at a slower rate than leaves in air, as seen previously for pathogeninduced 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

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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

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 chlorophyll 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

Fig. 3A±C. E€ects of light and darkness on the appearance of ELIPTOB 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

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)

Discussion

Chl decreased in tobacco leaves (Fig. 2). The cDNAAFLP-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

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

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

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(Kolanus et al. 1987). The ELIP-TOB aligns well with segment I of the three potential transmembrane segments 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 Kloppstech 1984; Scharnhorst et al. 1985). The light requirement 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 domains 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 (Kloppstech 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 upregulated 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

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up-regulation of the ELIP-TOB mRNA, whereas retardation 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 syndrome. This conclusion is based on the absence of ELIPTOB 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 upregulated 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-Paraskevopoulou 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 senescence that also occur during chloroplast development or stress, e.g. modi®cations of pigment/membrane associations 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 pigmentcarrier 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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