| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online July 9, 2004; 10.1104/pp.104.041699 Plant Physiology 135:1565-1573 (2004) © 2004 American Society of Plant Biologists Spermidine Synthase Genes Are Essential for Survival of ArabidopsisDivision of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060–0810, Japan (A.I., Y.H., Y.K., T.T.); Environmental Biology Division and Biodiversity Conservation Research Project, National Institute for Environmental Studies, Tsukuba, Ibaraki 305–0053, Japan (T.M., M.T., H.S.); Department of Low-Temperature Sciences, National Agricultural Research Center for Hokkaido Region, Sapporo 062–8555, Japan (T.A.); Mitsui Plant Biotechnology Research Institute (disbanded in March 1999), Tsukuba, Ibaraki 305–0047, Japan (Y.S., D.S.); Kazusa DNA Research Institute, Kisarazu, Chiba 292–0812, Japan (T.K., D.S., S.T.); and Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo 113–8657, Japan (H.H.)
The cellular polyamines putrescine, spermidine, and spermine are ubiquitous in nature and have been implicated in a wide range of growth and developmental processes. There is little information, however, on mutant plants or animals defective in the synthesis of polyamines. The Arabidopsis genome has two genes encoding spermidine synthase, SPDS1 and SPDS2. In this paper, we describe T-DNA insertion mutants of both of these genes. While each mutant allele shows normal growth, spds1-1 spds2-1 double-mutant seeds are abnormally shrunken and they have embryos that are arrested morphologically at the heart-torpedo transition stage. These seeds contain significantly reduced levels of spermidine and high levels of its precursor, putrescine. The embryo lethal phenotype of spds1-1 spds2-1 is complemented by the wild-type SPDS1 gene. In addition, we observed a nearly identical seed phenotype among an F2 seed population from the cross between the spds2-1 allele and SPDS1 RNA interference transgenic lines. These data provide the first genetic evidence indicating a critical role of the spermidine synthase in plant embryo development.
Polyamines are ubiquitous nitrogen compounds that are generally recognized as being necessary for orderly patterns of growth and development in most organisms (Tabor and Tabor, 1984  ; Cohen, 1998). Due to their positive charge, polyamines interact with various macromolecules and membranes. They show specific and differential binding to phospholipids and affect membrane rigidity (Schuber, 1989). Polyamines promote polymerization of cytoskeletal components, influence DNA conformation and stability, and activate the ribosomal ternary complex during protein synthesis (Igarashi and Kashiwagi, 2000). In higher plants, extracellular polyamines may interact with negatively charged cell wall components (Mariani et al., 1989). Growth-promoting activity of polyamines in plants was first demonstrated for callus production in explants of Jerusalem artichoke tubers (Bagni, 1966). Since then many studies on plants have implicated polyamines in a variety of cellular processes, including response to stress, control of cell division, root formation, flowering, and retardation of senescence (Evans and Malmberg, 1989; Galston and Sawhney, 1990; Kumar et al., 1997; Walden et al., 1997; Bouchereau et al., 1999). Involvement of polyamines in the stabilization of thylakoid membranes has also been reported for some plant species (Besford et al., 1993). However, the mechanisms underlying regulation of intracellular levels of polyamines and their localization have not been elucidated.
While the biosynthesis of polyamines in most organisms is initiated by decarboxylation of Orn to form putrescine via Orn decarboxylase (ODC), plants and some microorganisms can generate putrescine from Arg, via Arg decarboxylase (ADC) and agmatine ureohydrolase or agmatine iminohydrolase (Fig. 1). The respective functions of the two pathways of putrescine biosynthesis via ODC and ADC are still not clear. The genome of Arabidopsis contains no gene sequence for ODC (Hanfrey et al., 2001
Genetic analyses have revealed that the yeast spe3 mutant, which has no spermidine synthase activity, has an absolute requirement for spermidine or spermine for growth, while deletion of the SPE4 gene encoding spermine synthase has no effect on growth (Hamasaki-Katagiri et al., 1997 , 1998). In Escherichia coli, spermidine-deficient mutants are still able to grow at near-normal rates without exogenous polyamines (Xie et al., 1993). In mouse fibroblast cell cultures, disruption of a spermine synthase gene that causes a lack of spermine and a higher accumulation of spermidine had no significant effect on the growth of cells (Mackintosh and Pegg, 2000). In higher plants, disruption of the ACAULIS5 (ACL5) gene, which encodes spermine synthase in Arabidopsis, has been shown to result in a severely dwarfed phenotype (Hanzawa et al., 2000). Furthermore, our recent study has demonstrated that spermine is not essential for survival of Arabidopsis, at least under normal growth conditions (Imai et al., 2004). However, no direct genetic evidence indicating the significance of spermidine in plant cells has been obtained. In this paper, we show that the genes encoding spermidine synthase are essentially required for embryonic development of Arabidopsis.
Identification of T-DNA Insertion Mutants in SPDS1 and SPDS2
The Arabidopsis genome contains two genes encoding spermidine synthase, SPDS1 and SPDS2. These gene products have been shown to possess spermidine synthase activity (Hanzawa et al., 2002
Phenotype of the spds1 spds2 Double Mutant To determine whether the normal growth of each single mutant is attributable to the functional redundancy between SPDS1 and SPDS2, we made spds1-1 spds2-1 double mutants. Since SPDS1 and SPDS2 belong to the same linkage group (chromosome I), we first selected spds1-1/spds1-1 spds2-1/+ and spds1-1/+ spds2-1/spds2-1 plants in an F2 generation of the reciprocal cross between spds1-1 and spds2-1 homozygous mutants by PCR-based genotyping. We then examined the siliques of these self-pollinated F2 plants and found that there are aborted seeds randomly distributed throughout the silique (Fig. 3A). These seeds, which become dark brown and shrink as they mature, apparently fail to germinate. The segregation ratio of these abnormal seeds was approximately a quarter of the total F3 seeds harvested (Table I). We confirmed that the rest of the seeds show normal germination and growth. These normal seedlings were found to contain at least one copy of the wild-type SPDS1 or SPDS2 gene depending on the parental F2 genotype (Table I). No seedlings homozygous for both spds1-1 and spds2-1 alleles were obtained, suggesting that the double mutant is embryonically lethal. Similar results were obtained from the reciprocal cross between spds1-1 and spds2-2 homozygous mutants (data not shown).
To characterize the nature of the seed lethality, we examined the embryos in the developing F2 siliques under a microscope. Approximately one-quarter of the immature seeds contained embryos defective in development. Figure 3, B and C, show that while a normal embryo reaches the bent-cotyledon stage at 3 d after pollination, a defective embryo remains at the heart stage. The defective embryos were eventually arrested at the heart-torpedo transition stage in mature dry seeds (Fig. 3, D and E). PCR-based genotyping analysis revealed that these arrested embryos are spds1-1 spds2-1 double mutants (Fig. 3F). We further investigated the levels of putrescine, spermidine, and spermine in these embryos. Polyamines were extracted from dry seeds, dansylated, and analyzed by HPLC. The bound polyamine levels (perchloric acid [PCA]-insoluble fraction) in Arabidopsis seeds were just at the limits of our ability to detect them, and we therefore focused on the free and conjugated polyamines (PCA-soluble fraction; Fig. 4, A and B). In both free and conjugated forms, no significant difference was found between the wild type and each single mutant with respect to the levels of the three major polyamines. In contrast, the free spermidine and conjugated spermidine levels in the spds1-1 spds2-1 double-mutant seeds were decreased to 20.1% and 17.1% of those in the wild-type seeds, respectively. Spermine levels in spds1-1 spds2-1 seeds were also reduced. On the other hand, the free and conjugated putrescine levels in the double-mutant seeds were 17.0- and 4.8-fold greater than those in the wild-type seeds, respectively.
We performed a genetic complementation experiment with the wild-type SPDS1 gene. A genomic fragment encompassing the entire coding region of SPDS1 and its flanking 5' sequence was used for transformation of spds1-1/spds1-1 spds2-1/+ plants. We obtained spds1-1 spds2-1 double mutant plants that contained a wild-type SPDS1 transgene and exhibited normal development in the progeny of the transformants. The progeny of self-fertilized spds1-1 spds2-1 plants with one copy of the SPDS1 transgene showed a segregation ratio that was a good fit to the expected ratio of normal seeds:shrunken seeds (3:1; Table I).
To further confirm that the embryonic arrest is associated with a defect in spermidine synthesis, we examined the effect of suppression of SPDS1 expression by RNA interference (RNAi) analysis (Chuang and Meyerowitz, 2000
Physiological data obtained in various systems support the role of polyamines as regulators of cell proliferation and differentiation. Large amounts of spermidine and spermine accumulate during the transition from the G1 to the S phase of the cell cycle in both plants and animals (Fuller et al., 1977 ). The fact that inhibition of polyamine biosynthesis results in cell cycle arrest at the G1 phase suggests the involvement of polyamines in DNA synthesis (Kramer et al., 2001; Chattopadhyay et al., 2002). In tobacco (Nicotiana tabacum) ovary tissues, dramatic synchronous increases in polyamine titers and in rates of polyamine biosynthesis, macromolecular synthesis, and cell division occur for a few days after fertilization (Slocum and Galston, 1985). Taken together with these findings, our observation that spds1-1 spds2-1 and spds1-1 spds2-2 embryos did not develop beyond the torpedo stage indicates that spermidine synthase has an essential role in cell proliferation during embryogenesis.
Since a T-DNA in spds1-1 is located within the SPDS1 coding sequence for the large C-terminal catalytic core domain (Korolev et al., 2002
On the other hand, a markedly increased level of putrescine in the double mutant embryo may be due to the blockage of its conversion to spermidine. This raises the possibility that embryonic arrest in the double mutant is associated with toxicity of overaccumulated putrescine. Previous studies have shown that overexpression of oat ADC in tobacco leads to abnormal phenotypes such as short internodes, thin stems and leaves, leaf necrosis, and reduced root growth, the severity of which is correlated with putrescine content (Masgrau et al., 1997
The wild-type phenotype of spds1-1, spds2-1, and spds2-2 single-mutant plants can be interpreted by functional redundancy between SPDS1 and SPDS2. Both SPDS1 and SPDS2 transcripts are present in all organs and are present at higher levels in the root tissue than in other organs (Hanzawa et al., 2002
We used RNAi to examine whether the morphological defect of double-mutant embryos can also be realized by the suppression of SPDS1 gene expression in spds2-1. The construct was made to produce a double-stranded RNA containing the SPDS1 promoter sequence for triggering transcriptional gene silencing by de novo methylation of the target promoter (Mette et al., 2000
In conclusion, the isolation of SPDS1 and SPDS2 T-DNA insertion mutants and the construction of their double mutants enabled us to prove the importance of these genes in plant survival. A previous study demonstrated that the stem elongation of acl5 mutants could be restored in a heat shock-dependent manner by introducing heat shock-inducible ACL5 cDNA (Hanzawa et al., 2000
Plant Growth Conditions The Columbia (Col-0) ecotype of Arabidopsis (L.) Heynh was used as the wild type. Plants were grown under continuous fluorescent light of 120 µmol photons m–2 s–1 at 22°C on rock-wool bricks supplemented with vermiculite or on 0.8% (w/v) agar plates containing Murashige and Skoog salts (pH 5.8) and 3% Suc after surface sterilization of seeds.
The spds1-1 and spds2-1 mutant lines were obtained by screening a DNA pool of the Arabidopsis (ecotype Col-0) T-DNA insertion lines deposited at the Kazusa DNA Research Institute. PCR reactions included gene-specific primers, S1F (5'-CATTTCTCGGAGATATTCACCAG-3') and S1R (5'-CCTCCAGATTAGTTTTCTTTCCC-3') for SPDS1 and S2F (5'-CTAATCTCTTACTCACTGTCTCTCT-3') and S2R (5'-CTAGTTGGCTTTCGAATCAATCACC-3') for SPDS2 in combination with a T-DNA left-border (LB) primer (5'-ATAACGCTGCGGACACATCTAC-3') and a right-border primer (5'-ATCCAGGCTTTGATAGTCA-3'). T-DNA insertion sites were determined by sequencing the resulting positive PCR fragments with the LB or right-border primer. Plant genotyping for spds1-1 and spds2-1 alleles was performed by PCR using the SPDS1 and SPDS2 gene-specific primers and the LB primer described above. An spds2-2 mutation was found in the searchable database of T-DNA insertion sequences established by the Salk Institute Genome Analysis Laboratory. The T-DNA Express database is accessible at http://signal.salk.edu/cgi-bin/tdnaexpress. DNA flanking the LB of the T-DNA was amplified with a T-DNA LB' primer (5'-ACGTCCGCAATGTGTTAT-3') and sequenced. Plant genotyping for the spds2-2 allele was performed by PCR using the SPDS2 gene-specific primers and the LB' primer described above.
Total RNA was extracted from 10-d-old seedlings grown on Murashige and Skoog agar plates according to the SDS-phenol method (Takahashi et al., 1992 For RNA gel-blot analysis, 10 µg of each total RNA sample was separated on 1.2% formaldehyde agarose gels and blotted onto Hybond-N+ membranes (Amersham, Buckinghamshire, UK). Gene-specific probes for SPDS1 and SPDS2 were prepared by PCR using Arabidopsis genomic DNA as a template with primer pairs, S1F' (5'-TTCGCCAAGAAGGTCATTGAGTCA-3') and S1R' (5'-TGATGTTAATGTTCTTGGTCTTCGG-3') for the SPDS1 3'-UTR, and S2F' (5'-TTTTGCTAAGAAGGTGATTGATTCG-3') and S2R' (5'-TATAACGTCAACGTCGACAATACC-3') for the SPDS2 3'-UTR. These fragments were 32P-labeled by random-primed synthesis (Takara). The blots were hybridized at 42°C for 16 h with a labeled probe in 50% (v/v) formamide, 10% (w/v) dextran sulfate, 1 M NaCl, and 1% SDS and then washed twice with 2 x SSC, 0.1% SDS at 65°C for 30 min, and once with 0.1 x SSC at room temperature for 5 min. The membranes were exposed to x-ray films at –80°C for 72 h.
Seeds of wild-type and spds1-1 spds2-1 double-mutant plants were harvested from siliques, fixed in ethanol:acetic acid (6:1) solution for 4 h and washed successively in 90% and 70% (v/v) ethanol for 30 min each. Then the seeds were cleared with 72.7% (w/v) chloral hydrate in 50% (w/v) glycerol for 4 h prior to microscopy. Embryos were visualized using a Zeiss (Jena, Germany) LSM410 invert Laser Scan Microscope equipped with Nomarski optics. Mature embryos in dry seeds were dissected using fine forceps after the seeds were imbibed.
In order to extract polyamines, seeds (10 mg) were homogenized in 0.5 mL of 5% (w/v) PCA containing 1 nmol of 1,6-hexanediamine as an internal standard. After centrifugation, the supernatant was preserved and the pellet was resuspended in 5% PCA after several washes with the same solution. Aliquots of acid-soluble and acid-insoluble fractions, containing free plus conjugated polyamines and bound polyamines, respectively, were subjected to hydrolysis in 6 M HCl at 110°C for 18 h to convert the conjugated and bound forms to free form. After the hydrolyzate was taken to dryness in vacuum at 70°C, the residues were dissolved in 500 µL of 5% PCA. Aliquots (100 µL) were added to 200 µL of saturated sodium carbonate and 200 µL of dansyl chloride (5 mg mL–1 acetone). After brief vortexing, the mixture was incubated in darkness at 30°C for 16 h. Excess dansyl reagent was inactivated by the addition of 50 µL of 0.9 M Pro. Dansylated polyamines were extracted in 0.5 mL toluene, dried in Speed-Vac concentrator (Savant, Holbrook, NY), redissolved in 50 µL of methanol, and analyzed by HPLC using a Wakosil-II 5C18 HG reverse phase column (particle size 5 µm; 4.6 x 150 mm; Wako, Osaka). Portions (20 µL) of the polyamine fractions were applied to the column and eluted with a programmed methanol to water solvent gradient, changing from 55% to 85% over 15 min at a flow rate of 0.8 mL min–1. Elution was complete after 15 min. Polyamines were quantified by a fluorescence detector set at excitation and emission wavelengths of 365 and 510 nm, respectively. Conjugated polyamine content was calculated by subtracting free polyamine content from total acid-soluble polyamine content. Results were standardized with equimolar (0.5 nmol) mixtures of dansylated polyamines.
A 3,854-bp genomic fragment containing the SPDS1 gene was amplified by PCR using the forward primer cF (5'-CTATCGATGATGCGGATGCG-3') at 1,049 bp upstream of the SPDS1 ATG start codon and the reverse primer cR (5'-TGGAATTCGGTCTTGCGACAAGGA-3') at 490 bp downstream of the stop codon. The underlined sequences are endogenous ClaI and engineered EcoRI restriction sites, respectively. The fragment was then cloned into the pBI101.2 vector (CLONTECH, Palo Alto, CA) at ClaI-EcoRI sites, resulting in a cSPDS construct.
To make a hairpin RNA construct for SPDS1, a 0.9-kb fragment (from –564 to +335 relative to the transcriptional initiation site) and a 1.4-kb fragment (from –564 to +881) of the SPDS1 gene were each amplified by PCR from genomic DNA with the following primer pairs: S1Fi (5'-AAGCTTCTTCGTTTAAATGCCTTCC-3')/S1Ri (5'-GGATCCACCCAGGAATAACAGTG-3') and S1Fi / S1R'i (5'-GGATCCTACTTTACCGGAGAAGAA-3'), respectively. The underlined sequences are additional HindIII and BamHI restriction sites. These amplified fragments were separately cloned into the pGEM-T Easy vector (Promega, Madison, WI) to generate pSPDS1a and pSPDS1b, respectively. The SpeI-BamHI fragment of pSPDS1a and the BamHI-SacI fragment of pSPDS1b were successively transferred into XbaI-BamHI and BamHI-SacI sites downstream of the CaMV35S promoter of a binary vector, pBI121 (CLONTECH), to remove the
Transformation of Arabidopsis was carried out by the floral dip method (Bechtold and Pelletier, 1998 Received February 26, 2004; returned for revision April 26, 2004; accepted April 27, 2004.
1 Present address: Research-domain 37, Toyota Central R&D Lab., Inc., Aichi 480–1192, Japan. 
2 Present address: John Innes Centre, Colney, Norwich NR4 7UH, UK.
3 Present address: Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853.
4 Present address: Division of Biological Sciences, The University of Tokyo, Tokyo 113–0033, Japan.
5 Present address: Department of Biology, Faculty of Science, Okayama University, Okayama 700–8530, Japan. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.041699. * Corresponding author; e-mail perfect{at}cc.okayama-u.ac.jp; fax 81–86–251–7858.
Alabadí D, Carbonell J (1999) Differential expression of two spermidine synthase genes during early fruit development and in vegetative tissues of pea. Plant Mol Biol 39: 933–943[CrossRef][ISI][Medline] An YQ, McDowell JM, Huang S, McKinney EC, Chambliss SC, Meagher RB (1996) Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. Plant J 10: 107–121[CrossRef][ISI][Medline] Bagni N (1966) Aliphatic amines and a growth factor of coconut milk as stimulating cellular proliferation of Helianthus tuberosus (Jerusalem artichoke) in vitro. Experientia 22: 732–733 Bechtold N, Pelletier G (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82: 259–266[Medline] Besford RT, Richardson C, Campos JL, Tiburcio AF (1993) Effect of polyamines on stabilization of molecular complexes in thylakoid membranes of osmotically stressed oat leaves. Planta 189: 201–206 Bouchereau A, Aziz A, Larher F, Martin-Tanguy J (1999) Polyamines and environmental challenges: recent development. Plant Sci 140: 103–125[CrossRef]
Chattopadhyay MK, Tabor CW, Tabor H (2002) Absolute requirement of spermidine for growth and cell cycle progression of fission yeast (Schizosaccharomyces pombe). Proc Natl Acad Sci USA 99: 10330–10334
Chuang C, Meyerowitz EM (2000) Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci USA 97: 4985–4990 Cohen S (1998) A Guide to the Polyamines. Oxford University Press, New York Evans PT, Malmberg RL (1989) Do polyamines have roles in plant development? Annu Rev Plant Physiol Plant Mol Biol 40: 235–269[ISI] Fuller DJ, Gerner EW, Russell DH (1977) Polyamine biosynthesis and accumulation during the G1 to S phase transition. J Cell Physiol 93: 81–88[CrossRef][Medline]
Galston AW, Sawhney RK (1990) Polyamines in plant physiology. Plant Physiol 94: 406–410 Hamasaki N, Galston AW (1990) The polyamines of Xanthium strumarium and their response to photoperiod. Photochem Photobiol 52: 181–186[Medline] Hamasaki-Katagiri N, Katagiri Y, Tabor CW, Tabor H (1998) Spermine is not essential for growth of Saccharomyces cerevisiae: identification of the SPE4 gene (spermine synthase) and characterization of a spe4 deletion mutant. Gene 210: 195–201[CrossRef][ISI][Medline] Hamasaki-Katagiri N, Tabor CW, Tabor H (1997) Spermidine biosynthesis in Saccharomyces cerevisiae: polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase). Gene 187: 35–43[CrossRef][ISI][Medline] Hanfrey C, Sommer S, Mayer MJ, Burtin D, Michael AJ (2001) Arabidopsis polyamine biosynthesis: absence of ornithine decarboxylase and the mechanism of arginine decarboxylase activity. Plant J 27: 551–560[CrossRef][ISI][Medline] Hanzawa Y, Imai A, Michael AJ, Komeda Y, Takahashi T (2002) Characterization of the spermidine synthase-ralated gene family in Arabidopsis thaliana. FEBS Lett 527: 176–180[CrossRef][ISI][Medline] Hanzawa Y, Takahashi T, Michael AJ, Burtin D, Long D, Pineiro M, Coupland G, Komeda Y (2000) ACAULIS5, an Arabidopsis gene required for stem elongation, encodes a spermine synthase. EMBO J 19: 4248–4256[CrossRef][ISI][Medline] Igarashi K, Kashiwagi K (2000) Polyamines: mysterious modulators of cellular functions. Biochem Biophys Res Commun 271: 559–564[CrossRef][ISI][Medline] Imai A, Akiyama T, Kato T, Sato S, Tabata S, Yamamoto KT, Takahashi T (2004) Spermine is not essential for survival of Arabidopsis. FEBS Lett 556: 148–152[CrossRef][ISI][Medline] Korolev S, Ikeguchi Y, Skarina T, Beasley S, Arrowsmith C, Edwards A, Joachimiak A, Pegg AE, Savchenko A (2002) The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor. Nat Struct Biol 9: 27–31[CrossRef][ISI][Medline]
Kramer DL, Chang BD, Chen Y, Diegelman P, Alm K, Black AR, Roninson IB, Porter CW (2001) Polyamine depletion in human melanoma cells leads to G1 arrest associated with induction of p21WAF1/CIP1/SDI1, changes in the expression of p21-regulated genes, and a senescence-like phenotype. Cancer Res 61: 7754–7762 Kumar A, Altabella T, Taylor MA, Tiburcio AF (1997) Recent advances in polyamine research. Trends Plant Sci 2: 124–130 Mackintosh CA, Pegg AE (2000) Effect of spermine synthase deficiency on polyamine biosynathesis and content in mice and embryonic fibroblasts, and the sensitivity of fibroblasts to 1,3-bis-(2-chloroethyl)-N-nitrosourea. Biochem J 351: 439–447 Mariani P, D'Orazi D, Bagni N (1989) Polyamines in primary walls of carrot cells: endogenous content and interactions. J Plant Physiol 135: 508–510 Masgrau C, Altabella T, Farrás R, Flores D, Thompson AJ, Besford RT, Tiburcio AF (1997) Inducible overexpression of oat arginine decarboxylase in transgenic tobacco plants. Plant J 11: 465–473[CrossRef][ISI][Medline] Mette MF, Aufsatz W, van der Winden J, Matzke MA, Matzke AJM (2000) Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J 19: 5194–5201[CrossRef][ISI][Medline] Noury M, Bassie L, Lepri O, Kurek I, Christou P, Capell T (2000) A transgenic rice cell lineage expressing the oat arginine decarboxylase (adc) cDNA constitutively accumulates putrescine in callus and seeds but not in vegetative tissues. Plant Mol Biol 43: 537–544[Medline] Panicot M, Masgrau C, Borrell A, Cordeiro A, Tiburcio AF, Altabella T (2002a) Effects of putrescine accumulation in tobacco transgenic plants with different expression levels of oat arginine decarboxylase. Physiol Plant 114: 281–287[Medline]
Panicot M, Minguet EG, Ferrando A, Alcázar R, Blázquez MA, Carbonell J, Altabella T, Koncz C, Tiburcio AF (2002b) A polyamine metabolon involving aminopropyl transferase complexes in Arabidopsis. Plant Cell 14: 2539–2551 Paszkowski J, Whitham SA (2001) Gene silencing and DNA methylation processes. Curr Opin Plant Biol 4: 123–129[CrossRef][ISI][Medline] Schuber F (1989) Influence of polyamines on membrane functions. Biochem J 260: 1–10[ISI][Medline] Sebela M, Radová A, Angelini R, Tavladoraki P, Frébort II, Pec P (2001) FAD-containing polyamine oxidases: a timely challenge for researchers in biochemistry and physiology of plants. Plant Sci 160: 197–207[Medline]
Slocum RD, Galston AW (1985) Changes in polyamine biosynthesis associated with postfertilization growth and development in tobacco ovary tissues. Plant Physiol 79: 336–343 Tabor CW, Tabor H (1984) Polyamines. Annu Rev Biochem 53: 749–790[CrossRef][ISI][Medline]
Takahashi T, Naito S, Komeda Y (1992) Isolation and analysis of the expression of two genes for 81-kilodalton heat shock proteins from Arabidopsis. Plant Physiol 99: 383–390 Urano K, Yoshiba Y, Nanjo T, Igarashi Y, Seki M, Sekiguchi F, Yamaguchi-Shinozaki K, Shinozaki K (2003) Characterization of Arabidopsis genes involved in biosynthesis of polyamines in abiotic stress responses and developmental stages. Plant Cell Environ 26: 1917–1926[CrossRef] Walden R, Cordeiro A, Tiburcio AF (1997) Polyamines: small molecules triggering pathways in plant growth and development. Plant Physiol 113: 1009–1013[CrossRef][ISI][Medline] Xie QW, Tabor CW, Tabor H (1993) Deletion mutations in the speED operon: spermidine is not essential for the growth of Escherichia coli. Gene 126: 115–117[CrossRef][Medline] This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
RESULTS
-glucuronidase gene and generate an SPDS1 RNAi construct (
