Isolation and characterization of an isocitrate lyase ...

JOURNAL OF PLANT PHYSIOLOGY

J. Plant Physiol. 157. 669-676 (2000) © Urban & Fischer Verlag http://wwvv.urbanfischer.de/journals/jpp

Isolation and characterization of an isocitrate lyase gene from senescent leaves of sweet potato (Ipomoea batatas cv. Tainong 57) Hsien-Jung Chen" Wen-Chi Hou 2, Wann-Neng Jane 1, Yaw-Huei Lin 1 *

1

Institute of Botany, Academia Sinica, Taipei, Taiwan 115, Republic of China

2

Graduate Institute of Pharmacognosy Science, Taipei Medical University, Taipei, Taiwan 105, Republic of China

Received May 22, 2000 . Accepted July 28, 2000

Summary Isocitrate lyase and malate synthase are two key enzymes of the glyoxylate cycle, and are associated with lipid degradation and gluconeogenesis in microorganisms, plants, nematodes, and animals. Although genes of isocitrate lyase have been cloned from seedling cotyledons of several plants, none were isolated from the senescent leaves. In this study, sweet potato yellow leaves with disassembled chloroplast thylakoids and remarkable osmiophilic globule accumulation in stroma kb fuliwere used as material. Using both PCR-based subtractive hybridization and RACE PCR , a 2 .1 length spieL cDNA was cloned. The open reading frame contained 1728 nucleotides (576 amino acids) and exhibited more than 75 % sequence identity with plant cotyledon isocitrate Iyases of tomato, upland colton, cucumber, castor bean, rape, soybean, and loblolly pine. The spieL-encoded protein contained the Leu-169, Lys-170, Pro-171 (LKP) and Thr-210, Lys-211, Lys-212 (TKK) motifs, with a reported substrate binding domain function, as well as a putative peroxisomal targeting signal (PTS) Ala-Arg-Met (ARM) tripeptide at the C-terminus. Its mRNA accumulated exclusively in the senescing and completely yellow leaves, but not in the green leaves, roots, or stems. Hence, the sweet potato spieL is the first isocitrate lyase isolated from senescent leaves. The data may provide molecular evidence to support the notion that glyoxylate cycle, a metabolic pathway utilized in cotyledons for postgerminative growth of oilseeds, is also involved in lipid degradation and gluconeogenesis of senescent leaves. Key words: sweet potato - isocitrate lyase - glyoxylate cycle - leaf senescence - peroxisomal targeting signal

Introduction Leaf senescence is the final stage of leaf development, and has been considered as a type of programmed celi death. It is not, however, simply a degenerative process, but also a • E-mail correspondingauthor:[email protected]

recycling one in which nutrients are translocated from the senescent celis to young leaves, developing seeds, or storage tissues (Buchanan-Woliaston 1997). In a defined order, leaf celis undergo highly coordinated changes in structure , metabolism, and gene expression during senescence . The earliest and most significant change in celi structure is the breakdown of the chloroplast, which contains up to 70 % of 0176-1617/00/157/06-669 $1500/0

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the leaf protein (Makino and Osmond 1991). Metabolically, the carbon assimilation (photosynthesis) is replaced by catabolism of chlorophyll and macromolecules, such as proteins, membrane lipids, and RNAs so that some of the released nutrients can be recycled. Thus, remarkable osmiophilic globule accumulation in the chloroplast stroma following the thylakoid disassembly could be observed (Barton 1966, Bulter 1967). The membranes of plant cells constitute a valuable store of lipid molecules, which can be mobilized and used by the senescing leaves. It appears that the membranes of the cells, including the thylakoid membranes, are metabolized to provide energy for the senescence process (Wanner et al. 1991). For lipid degradation and gluconeogenesis, this requires the cooperation of several organelles, including glyoxysomes and mitochondria. The lipids are first metabolized into fatty acids, which then enter into the glyoxysomes and are degraded into acetyl-CoA by ~-oxidation. Through the glyoxylate cycle, succinate is generated from acetyl-CoA. It then leaves the glyoxysomes and enters into mitochondria for malate biosynthesis. The malate is utilized for gluconeogenesis in cytosol (Escher and Widmer 1997). Huang et al. (1983) reported that plant peroxisomes could be divided into leaf-type peroxisomes, glyoxysomes, root peroxisomes, and unspecialized peroxisomes. In cotyledons of fatty seedlings, glyoxysomes are replaced by leaf-type peroxisomes during greening (Huang et al. 1983). Leaf-type peroxisomes could possibly exhibit a functional transition to glyoxysomes during leaf senescence (Gut and Matile 1988). In addition to the characteristic peroxisomal enzymes, glyoxysomes contain enzymes of the fatty acid ~-oxidation and the glyoxylate cycle, which was originally identified in microorganisms as the pathway allowing growth on acetate. The glyoxylate cycle was also shown to operate in endosperm tissues of castor oilseeds (Kornberg and Beevers 1957). The malate synthase and isocitrate lyase are two key enzymes of the cycle, and have been shown to increase in senescing leaves of barley (Gut and Matile 1988), rice, and wheat (Pistelli et al. 1991), and senescent cotyledons of cucumber (McLaughlin and Smith 1995) and pumpkin (De Bellis and Nishimura 1991). Isocitrate lyase genes have been isolated previously from the seedling cotyledons of tomato (unpublished, accession number U 18678), cotton (Turley et al. 1990), cucumber (Reynolds and Smith 1995), loblolly pine (Mullen and Gifford 1997), pumpkin (Mano et al. 1996), castor bean (Beeching and Northcote 1987), soybean (unpublished, accession number L02329), and rape (Zhang et al. 1993). However, the senescence-associated isocitrate lyase gene has not yet been cloned from the senescent leaves. Here we report, for the first time, that a full-length cDNA encoding a putative isocitrate lyase with a tissue-specific expression in senescent leaves of sweet potato was isolated. Our results may provide evidence at the molecular level to support the possible operation of the glyoxylate cycle in senescent leaves.

Materials and Methods Plant materials and chemicals The storage roots of sweet potato (Ipomoea batatas cv. Tainong 57) were purchased from a local supermarket and grown in the greenhouse. Plantlets grown from the storage roots were used as test materials. Fully expanded green leaves near the top of stems and leaves with different levels of senescence were collected separately, frozen with liquid N2 , and kept in a -70"C freezer until use.

Electron microscopy Fully expanded green leaves and completely yellow leaves of sweet potato were fixed in 0.1 mol/L sodium phosphate buffer pH 7.2 containing 2.5 % glutaraldehyde and 4 % paraformaldehyde at room temperature for 3 h. The samples were rinsed three times before postfixed in the same buffer plus 1 % OS04 at room temperature for another 3 h. After three 20-min rinses in buffer, the materials were dehydrated using an acetone series, infiltrated and embedded with Spurr's resin, and sectioned with an Ultracut E ultramicrotome. Thin sections (60-90nm) were stained with uranyl acetate and lead citrate (Reynolds 1963), and observed with a Philips CM 100 transmission electron microscope.

Measurement of pigments For quantitative analysis of pigment contents, the fully expanded green leaves and senescing leaves of sweet potato were collected separately, ground in liquid N2 , and extracted with 2.5 mmol . L -1 sodium phosphate buffered 80 % acetone (pH 7.8) in a 1 : 4 (w : v) ratio at room temperature on an orbital shaker for 5 h. The absorbance of supernatants after centrifugation at 3,000 xg at room temperature for 20 min were measured at wavelengths of 663.8 nm, 646.8 nm, and 470 nm, respectively. Quantitative values of pigments for aqueous 80 % acetone extracts were calculated from the absorbance data, according to the report of Lichtenthaler (1987).

PCR-based subtractive hybridization and RACE PCR Total RNAs were isolated separately from the fully expanded green leaves and senescing leaves of sweet potato, basically according to the method of Sambrook et al. (1989). After separation with a mRNA purification kit (Promega), the total RNAs were vacuum-dried, then resuspended in a small volume of DEPC-H 2 0. The cDNAs were synthesized and used for cloning the differentially expressed cDNAs using a PCR-based subtractive hybridization kit (Clontech), following the protocols supplied by the manufacturer. The double-strand cDNAs of senescing leaves were subtracted by that of fully expanded green leaves, then ligated to the pGEM-T vector for E. coli DH5a competent cell transformation. Recombinant plasmids were isolated for DNA sequencing using an ASI PRIZM 337 DNA Sequencer. Nucleotide sequence data were analyzed using the Genetics Computer Group (GCG) programs. The RACE PCR method with the Marathon cDNA amplification kit (Clontech) was used to isolate the 5' and 3' ends of important cDNA inserts, according to the protocols provided by the manufacturer.

Isocitrate lyase of senescent leaves

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Northern blot hybridization For Northern blot hybridization, 30l-lg of total RNAs isolated from roots, stems, fully expanded green leaves, and senescing leaves were applied to a formaldehyde denaturing gel, then transferred to an Amersham H-bond N+ nylon membrane after electrophoreSiS, according to the methods of Molecular Cloning (Sambrook et al. 1989). The partial cDNA inserts containing putative isocitrate lyase (Y503), Rubisco small subunit (G317), and 26S ribosomal RNA (Y494), respectively, were labelled with digoxigenin-11-dUTP nucleotide as probes for Northern blots and Southern blots with CSPD substrate (Boehringer Mannheim).

Southern blot hybridization Young (not expanded) leaves of sweet potato were harvested and ground in liquid N2 . The powder was transferred to a centrifuge tube , mixed gently and thoroughly with CTAB buffer (2% CTAB, 1.4moIL- 1 NaCI, 20mmoieL -1 EDTA. 0.2% p-mercaptoethanol, and 100mmoleL-1 Tris-HCI pH 8.0) in a 20 : 1(w : v) ratio, and kept at 60 ·C in a water bath for about 30 min before adding an equal volume of chloroform and being centrifuged at 5,OOOxg at room temperature for 10min. The supernatant was transferred to a new centrifuge tube and the chloroform extraction step was repeated until the interface was clear. The total nucleic acid after precipitation with an equal volume of isopropanol was redissolved in sterile water, digested with restriction enzymes (EcoRI, Hindlll, and Xbal), and separated on 0.8 % agarose gels. The method for DNA transfer onto a hybond W nylon membrane (Amersham) basically followed the protocol of Molecular Cloning (Sambrook et al. 1989). The method for preparing the dig-labelled probe spieL was as described earlier.

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Results Chloroplast morphology of green leaves and yellow leaves The morphologies of subcellular organelles from fully expanded green leaves and completely yellow leaves were compared with electron microscopy. The peroxisomes of both green leaves and yellow leaves exhibited similar morphology (Fig. 1). In green leaves, the chloroplasts contained intact thylakoids and grana (Fig. 1 A). These structures, however, were disassembled during leaf senescence, and a remarkable increase in size and amount of osmiophilic globules in the stroma of completely yellow leaves was observed (Fig. 1 B). The endoplasmic reticulum and Golgi apparatus were also disassembled in yellow leaves (data not shown). Similar observation has been reported for senescing cucumber cotyledons (Bulter 1967) and completely yellow leaves of PhaseoIus vulgaris (Barton 1966). Senescence is in general a developmentally regulated program, and can be divided into reversible and irreversible phases in wheat leaves (Wittenbach 1977) and cucumber cotyledons (McLaughlin and Smith 1995). In senescing cucumber cotyledons, the isocitrate lyase and malate synthase were synthesized during the terminal irreversible phase when endoplasmic reticulum and

Figure 1. The cellular fine structures of both peroxisome and chloroplast of fully expanded green leaves and completely yellow leaves of sweet potato. (A) Green leaf. (B) completely yellow leaf. The arrowhead indicates the osmiophilic globule in chloroplast. Ch = chloroplast; G = granum; P = peroxisome; T = thylakoid.

Golgi apparatus were disassembled following the thylakoid breakdown (McLaughlin and Smith 1995). Since the completely yellow leaves with disassembled chloroplast thylakoids were possibly at a terminal irreversible phase, they were used to clone the glyoxysomal isocitrate lyase or malate synthase cDNAs.

DNA and amino acid sequences of sweet potato splCL Using PCR-based subtractive hybridization and RACE PCR (Clontech) methods, more than 500 recombinant colonies were isolated and analyzed from sweet potato senescent leaves. After DNA sequence comparison, using GCG program, a clone Y503 containing a cDNA insert (ca. 500 bp in length) was identified to encode a partial putative glyoxysomal isocitrate lyase protein. With the RACE PCR technique,

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Figure2. DNA and amino acid sequences of sweet potato spieL. The spieL open reading frame contains 1728 nucleotides and encodes 576 amino acids. The LKP and TKK motifs constitute the putative substrate-binding domain, and ARM is the putative peroxisomal targeting signal. The * indicates the stop codon of spieL protein.

Isocitrate lyase of senescent leaves

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Figure3. Alignment of isocitrate lyase C -terminal amino ac id sequences among different species. Sweet potato (ac cession #AF243525), castor 96) , rape (accession #JQ 1105), cucumber (accession #S53505), pumpkin (accession bean (accession #806274), c otton (accession #S 10771 and 8 10 3 #078256), loblolly pi ne (ac cession#T09779), soybean (accession # T07632and T07631), and tomato( accession #T60353). Twoconserved putative per0xi so ma
targeting signals, alanine-arginine - methionine (ARM) and serine-lysine - leucine (SKL), were found at the C-termini of isocitrate Iyases

a full -length splCL cDNA was cloned and sequenced (Fig. 2). It contained 1728 nucleotides (576 amino acids) in the open reading frame, and exhibited high amino acid sequence identities with plant isocitrate Iyases of tomato (Lycopersicon esculentum, accession number T60353, 87.3 %), upland cotton (Gossypium hirsutum, accession number 810771, 85.6 %), cucumber (Cucumis sativus, accession number 853505,84.7%), castor bean (Ricinus communis var. zanzibarensis, accession number 806274 , 83.2 %), rape (Brassica napus, accession number JQ1105, 81 .6 %), soybean (Glycine max, accession number T07631 and T07632; 81.4 % and 81.5 %, respectively), loblolly pine (Pinus taeda, accession number T09774 and T09779; 77.4 % and 75.6 %, respectively) , pumpkin (Cucurbita sp . cv. Kurokawa Amakuri Nankin, accession number 078256, 76.1 %), and inky cap (Coprinus cinereus, accession number JC6182, 56.7 %). Much lower sequence identities were also found with the fungal and bacterial isocitrate Iyases of Emericella nidulans (accession number 826857, 55.7%) , Neurospora crassa (accession number 826858, 54.2 %), yeast (Candida tropicalis, accession number JX0105 , 53.2 %, Saccharomyces cere visiae, accession number 822386 and 852819; 51.1 % and 41 %, respectively, Yarrowia lipolytica, accession number 839953 , 50 %), Mycobacterium leprae (accession number 877654,36.6 %), Escherichia coli (accession number 805692, 32 %), and Corynebacterium glutamicum (accession number 140713,30.9%). The splCL-encoded protein (Figs. 2, 3) contained the Leu169, Lys-170, Pro-171 (LKP) and Thr-210, Lys-211 , Lys-212 (TKK) motifs, with a reported substrate binding domain function for isocitric acid (Mano et al. 1996) near the N-terminal portion, as well as a putative peroxisomal targeting signal ARM at the C-terminus. The C-terminal ARM tripeptide, like the 8RM tripeptide , belongs to the 8KL-like motif, and has been demonstrated as a peroxisomal targeting signal for protein import into peroxisomes in higher plants (Olsen et al. 1993, Trelease et al. 1994, 1996). The data suggests a possible glyoxysomal localization for the ARM-containing splCL protein .

splCL is a senescence-associated gene in leaves Gene expression of splCL was studied with Northern blot hybridization (Fig . 4). The contents of cellular pigments such as carotenoids , chlorophylls a and b were used as markers of leaf senescence. Three cDNAs encoding the putative 268 ribosomal RNA, glyoxysomal isocitrate lyase, and Rubisco small subunit, respectively, were labelled with digoxigenin-11dUTP and used as probes. Leaves were divided into three stages based on their cellular pigment contents. The 80 was the stage of fully expanded green leaves with chlorophylls assigned as a standard of 100 %. 81 was the stage of senescing leaves with ca. 25 % chlorophylls , and the stage of completely yellow leaves was classified as 82 with less than 5 % chlorophylls. The relationship between pigment content and mRNA accumulation was shown in Figure 4. The cellular pigment contents (Fig. 4 A) and the Rubisco small subunit mRNA (Fig. 4 B) exhibited a parallel reduction. In completely yellow leaves, the pigment content was less than 5 % of that of fully expanded green leaves, and the Rubisco small subunit mRNA was not detectable using Northern blot hybridization (Fig . 4B). The splCL mRNA was not detected in fully expanded green leaves (SO), however, it increased remarkably in the senescing (81) and completely yellow (82) leaves. The bands of isocitrate lyase detected on 81 and 82 lanes were not exactly on the same line (Fig . 4B), possibly attributable to an electrophoretic technique problem or uneven agarose gel preparation. For tissue specific expression, splCL mRNA was detected exclusively in senescent leaves but not in the green leaves, roots , and stems (Fig. 5). The Rubisco small subunit mRNA was detected only in leaves, with a decreasing mode during senescence. The 268 ribosomal RNA was found relatively constant for all stages and tissues studied (Figs. 4 B, 5). We conclude that splCL is a senescence-associated gene with tissue specificity in senescing and completely yellow leaves. The genomic DNA was digested with restriction enzymes for 80uthern blot hybridization with the splCL probe (Fig. 6). There were one (ca . 8kb) , one (ca. 2.3kb) and two (ca. 10 kb

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leaves (81) of sweet potato. Total RNAs isolated from root, stem, green leaves, and senescing leaves were analyzed with Northern blot hybridization using probes of spieL (isocitrate lyase), 268 rRNA (268 ribosomal RNA), and Rubisco 88 (Rubisco small subunit), as described in Materials and Methods.

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564Discussion spieL (Fig. 2) showed more than 75 % amino acid sequence identities with those reported plant isocitrate Iyases (Beeching and Northcote 1987, Turley et al. 1990, Zhang et al. 1993,

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Figure6. Southern blot hybridization of sweet potato spieL gene. The genomic DNA of sweet potato was digested with different restriction enzymes (EcoRI, Hindlll, or Xbal), and detected with spieL probe, as described in Materials and Methods.

Isocitrate lyase of senescent leaves Reynolds and Smith 1995, Mano et al. 1996, Mullen and Gifford 1997}. It has long been postulated that proteins imported into peroxisomes require peroxisomal targeting signals possibly located at the C-terminal, the N-terminal , or the internal portions of the proteins (Purdue and Lazarow 1994). splCL did contain a C-terminal ARM tripeptide motif similar to that of the other isocitrate Iyases (castor bean , cotton , soybean, and tomato; Figs 2 and 3). Whether the ARM tripeptide motif of splCL could function as a peroxisomal targeting signal in senescent leaves needs further investigation. Plant peroxisomes are in general divided into glyoxysomes, leaf-type peroxisomes, root nodule peroxisomes, and unspecialized peroxisomes (Huang et al. 1983). There were no obvious morphological differences between the leaf-type peroxisomes and glyoxysomes. Olsen et al. (1993) demonstrated that a glyoxysomal isocitrate lyase of Brassica napus L. could be imported into both leaf-type and root peroxisomes of transgenic Arabidopsis plants. The results suggest that the import of proteins into peroxisomes is in general not selective and does not playa determinative role in specifying organelle function. Different peroxisom es are possibly formed through a common mechanism, and each functional class is determined by the expression of a unique set of genes (Olsen 1995). Therefore, the glyoxysomes could exhibit functional transition into leaf-type peroxisome in sunflower cotyledons during greening, with a sharp decline of glyoxysomal isocitrate lyase activity (Franzisket and Gerhardt 1980). From Figure 1, similar morphologies were found for peroxisomes from both green leaves and yellow leaves of sweet potato. However, splCL mRNA accumulated specifically in senescing leaves and completely yellow leaves (Figs. 4 B , 5). These results agree with previous reports (Olsen et al. 1993, Olsen and Harada 1995) and suggest that splCL is a senescence-associated gene, which is likely activated during the functional transition of leaf-type peroxisomes to glyoxysomes. The chloroplast morphology of sweet potato leaf showed drastic changes during senescence. The chloroplast thylakoids were disassembled and increased numbers of osmiophilic globules were found in stroma of completely yellow leaves (Fig. 1). Similar morphological changes have been reported for senescing cucumber cotyledons (Bulter 1967) and completely yellow leaves of Phaseolus vulgaris (Barton 1966). Ikeda and Ueda (1964) suggest that these osmiophilic globules were possibly formed from the disassembled chloroplast thylakoid membranes. It was characterized as a functional transition of leaf-type peroxisomes to glyoxysomes, whose proposed role is to metabolize the products of thylakoid lipid breakdown (Gut and Matile 1988, 1989) and subsequent gluconeogenesis (Wanner et a1.1991) via glyoxylate cycle. The enzymatic activities of glyoxysomal isocitrate lyase and malate synthase have been detected in senescent leaves of barley (Gut and Matile 1988), rice, and wheat (Pistelli et al. 1991), and senescing pumpkin cotyledons (De Bellis and Nishimura 1991). Isolation of splCL (Fig. 2) and its mRNA accumu-

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lation specifically in senescing leaves (Figs. 4, 5) agree with these reports. The data may provide molecular evidence to support the synthesis of isocitrate lyase during functional transition of leaf-type peroxisomes to glyoxysomes in senescent leaves. Whether the isocitrate lyase acts directly on the products of thylakoid membrane breakdown or disassembled endomembrane systems, as suggested by Gut and Matile (1988, 1989), is still unclear and requires more data to address the issue. We conclude here that a senescence-associated isocitrate lyase gene with a putative glyoxysomal targeting signal ARM was isolated from completely yellow leaves of sweet potato. Production of polyclonal antibody of the putative splCL isocitrate lyase protein, and transient and transgenic expression of splCl)reporter gene fusion proteins will be used in the future in order to identify the subcellular organelle targeting and the possible targeting signals of the splCL-encoded protein .

References Barton R (1966) Fine structure of mesophyll cells in senescing leaves of Phaseo/us. Planta 71: 314-325 Beeching JR, Northcote DH (1987) Nucleic acid (cDNA) and amino acid sequences of isocitrate lyase from castor bean. Plant Mol Bioi 8: 471-475 Buchanan-Wollaston V (1997) The molecular biology of leaf senescence. J Exp Bot 48: 181-199 Bulter RD (1967) The fine structure of senescing cotyledons of cucumber. J E xp B ot18: 535-543 De Bellis L, Nishimura M (1991) Development of enzymes of the glyoxylate cycle during senescence of pumpkin cotyledons. Plant Cell Physiol 32: 555-561 Escher CL, Widmer F (1997) Lipid mobilization and gluconeogenesis in plants: do glyoxylate cycle enzyme activities constitute a real cycle? A hypothesis. Bioi Chem 378: 803-813 Franzisket U, Gerhardt B (1980) Synthesis of isocitrate lyase in sunflower cotyledons during the transition in cotyledonary microbody function. Plant Physiol65 : 1081-1084 Gut H. Matile P (1989) Breakdown of galactolipids in senescent barley leaves. Bot Acta 102: 31-36 Gut H, Matile P (1988) Apparent induction of key enzymes of the glyoxylic acid cycle in senescent barley leaves. Planta 176: 548-550 Huang AHC, Trelease RN, Moore ST Jr (1983) Plant Peroxisomes. Academia Press, New York Ikeda T, Ueda RR (1964) Light and electron microscopic studies on the senescence of chloroplasts in Elodea leaf cells. Bot Mag 336 -341 Kornberg HL, Beevers H (1957) The glyoxylate cycle as a stage in the conversion of fat to carbohydrate in castor beans. Bioch im Biophys Acta 26: 531-537 Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Meth Enzymol 148: 350-382 Makino A, Osmond B (1991) Effects of nitrogen nutrition on nitrogen partitioning between chloroplasts and mitochondria in pea and wheat. Plant Physiol 96 355-362 Mano S, Hayashi M, Kondo M, Nishimura M (1996) cDNA cloning and expression of a gene for isocitrate lyase in pumpkin cotyledons. Plant Cell Physiol 37: 941 - 948

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McLaughlin JC, Smith SM (1995) Glyoxylate cycle enzyme synthesis during the irreversible phase of senescence of cucumber cotyledons. J Plant Physiol146: 133-138 Mullen RT, Gifford DJ (1997) Regulation of two loblolly pine (Pinus taeda L.) isocitrate lyase genes in megagametophytes of mature and stratified seeds and during postgerminative growth. Plant Mol Bioi 33: 593-604 Olsen LJ, Harada JJ (1995) Peroxisomes and their assembly in higher plants. Annu Rev Plant Physiol Plant Mol Bioi 46: 123-146 Olsen LJ, Ettinger WF, Damsz B, Matsudaira K, Webb MA, Harada JJ (1993) Targeting of glyoxysomal proteins to peroxisomes in leaves and roots of a higher plant. The Plant Cell 5: 941-952 Pistelli L, De-Bellis L, Alpi A (1991) Peroxisomal enzyme activities in attached senescing leaves. Planta 184: 151-153 Purdue PE, Lazarow PB (1994) Peroxisomal biogenesis: multiple pathways of protein import. J Bioi Chem 269: 30065-30068 Reynolds ES (1963) The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J Cell Bioi 55: 541-552 Reynolds SJ, Smith SM (1995) The isocitrate lyase gene of cucumber: isolation, characterization, and expression in cotyledons following seed germination. Plant Mol Bioi 27: 487-497

Sam brook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY Trelease RN, Choe SM, Jacobs BL (1994) Conservative amino acid substitutions of the C-terminal tripeptide (Ala-Arg-Met) on cottonseed isocitrate lyase preserve import in vivo into mammalian cell peroxisomes. Eur J Cell Bioi 65: 269-279 Trelease RN, Lee MS, Banjoko A, Bunkelmann J (1996) C-terminal polypeptides are necessary and sufficient for in vivo targeting of transiently-expressed proteins to peroxisomes in suspension-cultured plant cells. Protoplasma 195: 156-167 Turley RB, Choe SM, Trelease RN (1990) Characterization of a cDNA clone encoding the complete amino acid sequence of cotton isocitrate lyase. Biochim Biophys Acta 1049: 223-226 Wanner L, Keller F, Matile P (1991) Metabolism of radiolabelled galactolipids in senescent barley leaves. Plant Sci 78: 199-206 Witten bach VA (1977) Induced senescence of intact wheat seedlings and its reversibility. Plant Physiol 59: 1039-1042 Zhang JZ, Gomez-Pedrozo M, Baden CS, Harada JJ (1993) Two classes of isocitrate lyase genes are expressed during late embryogeny and postgermination in Brassica napus L. Mol Gen Genet 238: 177-184