JXB Advance Access originally published online on May 7, 2004
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Journal of Experimental Botany, Vol. 55, No. 401, pp. 1441-1443, June 1, 2004
© 2004 Oxford University Press
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Boron nutrition of cultured tobacco BY-2 cells. IV. Genes induced under low boron supply
Received 27 October 2003; Accepted 3 March 2004
Laboratory of Plant Nutrition, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
* To whom correspondence should be addressed. Fax:+81 75 753 6128. E-mail: matoh{at}kais.kyoto-u.ac.jp
Abbreviations: B, boron; GST, glutathione S-transferase; RG-II, rhamnogalacturonan II; TOGT, tobacco glucosyltransferase.
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Genes whose expression was up-regulated in low boron (B)-acclimated tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) cells, which had been selected under a low supply of B, were screened by the cDNA differential subtraction method. Thirteen genes were identified, including early salicylate-inducible glucosyltransferase, glutamine synthetase, glutathione S-transferase, and a pathogenesis-related protein, which might constitute a rescue system for oxidative damage. This indicates that B deficiency might impose cellular redox imbalance on the cells. Two of the 13 genes were induced within 30 min of B removal in the parent cells, indicating fast signal transfer from the cell walls to the cytoplasm.
Key words: Boron, boron deficiency, Nicotiana tabacum, oxidative stress, tobacco BY-2 cultured cells.
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Boron (B) is an essential microelement for higher plants. Although this was first demonstrated 80 years ago by Warington (1923), B function in plant metabolism is still under investigation (Parr and Loughman, 1983). The authors first isolated a B-polysaccharide complex in radish cell walls (Matoh et al., 1993), and the sugar moiety of the complex was revealed to be rhamnogalacturonan II (RG-II, Kobayashi et al., 1996). The B–RG-II complex crosslinks pectic polysaccharide chains and thus works to form a pectic network in the cell walls (Kobayashi et al., 1999). In suspension-cultured tobacco cells, nearly all the B is localized to the apoplast (Matoh et al., 1992), and complexes exclusively with the RG-II regions of pectic polysaccharides (Kobayashi et al., 1997). When culture media are deprived of B, cell propagation ceases immediately and cells die. The mechanisms underlying the various metabolic disorders caused by cell wall defects under B deficiency are unknown. To investigate these mechanisms, it is necessary to determine the earliest disorder induced by B deprivation in plant cells. The authors report the identification of genes that are expressed under low B supply in low B-acclimated cells and in B-deficient cells.
Tobacco (Nicotiana tabacum cv. BY-2) cell culture, B-free media preparation, and establishment of the low B-acclimated cell line were performed as described previously (Matoh et al., 1992). Genes preferentially expressed in the low B-acclimated cells were screened by the cDNA differential subtraction method (Diatchenko et al., 1996). Poly(A)+ RNAs were prepared from the 5-d-old low-B acclimated and control cells. cDNA synthesis and subtractive hybridization were performed using a PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. The subtracted cDNA pool, which was enriched for fragments specific to the acclimated cells, was ligated with a pT7Blue (Novagen, Madison, WI) T-vector and introduced into Escherichia coli DH5
cells. From the library, 280 clones were sequenced and approximately 240 non-redundant sequences were obtained. The specificity of their expression was examined by differential screening (Jin et al., 1997) and northern hybridization. Briefly, the inserts from the library were amplified by polymerase chain reaction (PCR) and arrayed manually on membranes, then hybridized with the forward- or reverse-subtracted cDNA pools labelled using the AlkPhos Direct system (Amersham Biosciences, Piscataway, NJ). Clones that only produced a strong signal with the forward-subtracted probes were further analysed by northern hybridization. The insert of each clone was labelled using digoxigenin-PCR labelling mix (Roche, Basel, Switzerland) and hybridized with the total RNAs prepared from the 5-d-old cells. Thirteen sequences were thus identified as representing low B-inducible genes.
Table 1 shows the results of a BLASTX search (Altschul et al., 1997) for these sequences. Some of the identified genes are general stress-responsive genes, such as early salicylate-inducible tobacco glucosyltransferase (Togt; L7), glutamine synthetase (O7), class tau glutathione S-transferase (GST; U2), and pathogenesis-related protein (X5). Togt was first identified as gene that is quickly induced by salicylate (Horvath and Chua, 1996), and its expression is also up-regulated in tobacco leaves acclimated to oxidative stress (Vranová et al., 2002). TOGT most likely catalyses the glucosylation of hydroxycoumarin scopoletin, which acts as an antioxidant (Chong et al., 2002). Tau GST catalyses the conjugation of endo- and exogenous electrophilic compounds to glutathione. The enzyme also displays glutathione-dependent peroxidase activity and non-catalytic carrier activity for phytochemicals such as hormones or flavonoids (Edwards et al., 2000). GSTs are considered to have a protective role against oxidative stress through glutathione-conjugating or peroxidase activity on reactive lipid peroxides (Edwards et al., 2000). The barley homologue of X5 encodes ß-1,3-glucanase (Jutidamrongphan et al., 1991) and might belong to pathogenesis-related protein PR2. Induction of PR2 is associated with the acquisition of oxidative stress tolerance (Mullineaux et al., 2000). Zhao et al. (1998) reported enhanced expression of the glutamine synthetase gene under oxidative stress conditions in Arabidopsis.
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The possible induction of the 13 genes was examined in B-deprived cells by northern hybridization. For this experiment, 5-d-old control cells were collected on a 60 µm nylon mesh and divided into two aliquots. Each aliquot was suspended in B-free or control (1 mg B l–1) media for 1 min and filtered. After another two cycles of washing, cells were transferred to corresponding B-free or control media and cultured. Two of the 13 genes were up-regulated by B deprivation (Fig. 1). The induction occurred during the cell washing process, which took approximately 30 min. This indicates that the absence of B in cell walls is detected by the cytoplasm very quickly.
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The induction of general stress-responsive genes under B deficiency suggests that the downstream effects of B deficiency are similar to those of other stress conditions (Iturbe-Ormaetxe et al., 1998; Prasad et al., 1994; Schützendübel and Polle, 2002; Shalata et al., 2001). That is, B deficiency is likely to induce oxidative damage to the cell. The observations that ascorbic acid and glutathione levels decrease dramatically under B deficiency (Cakmak and Römheld, 1997; Lukaszewski and Blevins, 1996) and that high light conditions exaggerate the symptoms of B deficiency (Cakmak et al., 1995; Tanaka, 1966) are consistent with this notion. The enhanced expression of TOGT, GST, a pathogenesis-related protein, and glutamine synthetase might constitute a rescue system against oxidative damage under B deficiency. Cakmak and Römheld (1997) reported that, based on the assumption that B binds to phenolic compounds to prevent their oxidation to toxic radical species, B deficiency causes oxidative damage to the cell. In cultured tobacco BY-2 cells, however, B is exclusively localized in the cell walls and a significant association of B with membranes or phenolic compounds has not been confirmed (Kobayashi et al., 1997). Therefore, the authors assume that oxidative damage in the absence of B is due to a redox imbalance that somehow occurs because of the impaired cell wall structure, and not due to the oxidized phenolic species, at least in the very beginning of the deficiency.
Further study is needed to determine the molecular species of reactive oxygen species in the B-deficient cells. The authors are currently performing an expressed sequence tag microarray analysis to identify initial metabolic disorders due to B deficiency.
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Acknowledgements |
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The authors thank Dr Daisaku Ohta, Graduate School of Agriculture, Osaka Prefectural University and Dr Masaharu Mizutani, Institute for Chemical Research, Kyoto University, for their advice and discussion. This study was supported financially in part by the Grand-in-Aid from the Japan Society for the Promotion of Science to MK (No. 14760037) and TM (No.13460031).
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