JXB Advance Access originally published online on November 29, 2004
Journal of Experimental Botany 2005 56(412):525-536; doi:10.1093/jxb/eri031
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RESEARCH PAPER |
Regulation of lysine catabolism in Arabidopsis through concertedly regulated synthesis of the two distinct gene products of the composite AtLKR/SDH locus
Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel
* To whom correspondence should be addressed. Fax: +972 8 9344181. E-mail: gad.galili{at}weizmann.ac.il
Received 5 April 2004; Accepted 17 September 2004
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Abstract |
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Lysine catabolism in plants is initiated by a bifunctional LKR/SDH (lysine-ketoglutarate reductase/saccharopine dehydrogenase) enzyme encoded by a single LKR/SDH gene. Yet, the AtLKR/SDH gene of Arabidopsis also encodes a second gene product, namely a monofunctional SDH. To elucidate the regulation of lysine catabolism in Arabidopsis through these two gene products of the AtLKR/SDH gene, an analysis was carried out on the effects of the hormones, abscisic acid and jasmonate, as well as various metabolic and stress signals, including lysine itself, on their mRNA and protein levels. The response of the two gene products to the various treatments was only partially co-ordinated, but the levels of the monofunctional SDH mRNA and protein were always in excess over their bifunctional LKR/SDH counterparts. These results suggest that lysine catabolism is regulated primarily by the first enzyme LKR, while the excess level of SDH enables efficient flux of lysine catabolism following the LKR step. Analysis of transgenic plants expressing ß-glucoronidase fusion constructs with the AtLKR/SDH and monofunctional AtSDH promoters demonstrated that transcriptional regulation contributes to the modulation of expression of the bifunctional LKR/SDH and monofunctional SDH gene products in response to hormonal and metabolic signals. To test whether the enhanced expression of the LKR/SDH gene under various hormonal and metabolic signals is correlated with enhanced lysine catabolism, wild-type Arabidopsis and a knockout mutant lacking lysine catabolism were exposed to abscisic acid and sugar starvation. Free lysine accumulated to significantly higher levels in this knockout mutant than in the wild-type plants.
Key words: ABA, catabolism, essential amino acids, jasmonate, lysine-ketoglutarate reductase, saccharopine dehydrogenase, stress, sugar starvation
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Lysine metabolism in plants is regulated both by the rate of its synthesis and catabolism (Galili, 2002
). Lysine catabolism operates via the 
-amino adipic acid pathway and is largely regulated by the first two enzymes of this pathway, namely lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH) (Arruda et al., 2000; Galili et al., 2001). These two enzymes are linked to each other on a single bifunctional protein encoded by a single LKR/SDH gene (Arruda et al., 2000; Galili et al., 2001). Although the reason for the linkage between the LKR and SDH enzymes is still not entirely understood, previous studies showed that LKR activity, can be modulated by interacting with the SDH domain via a mechanism that is dependent on the small linker region connecting these enzymes (Zhu et al., 2002). It has also been hypothesized that this linkage enables a highly regulated flux of lysine catabolism through LKR activity, apparently in response to physiological, metabolic, and developmental signals (Galili et al., 2001).
The regulation of lysine catabolism via SDH is also not simple, because the pH optimum of plant SDH enzymes is much above the physiological pH of the plant cell cytosol, where this enzyme is localized, rendering SDH an apparently inefficient enzyme in vivo (Arruda et al., 2000; Galili et al., 2001). Notably, the LKR/SDH gene of Arabidopsis also encodes an additional monofunctional SDH enzyme that is transcribed from a promoter located inside the coding region of the LKR/SDH gene and is translated by an in-frame internal ATG codon (Tang et al., 1997, 2000). It was also hypothesized that the combination of the bifunctional LKR/SDH and the monofunctional SDH enzymes enables both a highly regulated (via LKR activity of LKR/SDH) and efficient (via the linked plus the not-linked SDH enzymes) flux of lysine catabolism (Tang et al., 2000; Galili et al., 2001). This may, however, depend on the relative expression levels of the bifunctional LKR/SDH and monofunctional SDH enzymes. To address this issue, the relative expression levels of the Arabidopsis bifunctional LKR/SDH and monofunctional SDH mRNAs and polypeptides in response to various hormonal and metabolic signals were analysed here. This was achieved using transgenic Arabidopsis plants encoding a haemagglutinin (HA)-tagged LKR/SDH gene, as well as two ß-glucoronidase (GUS) reporter constructs containing one or other of the two promoters of this gene. The results show that the regulation of expression of these two gene products is partially co-ordinated, and the levels of monofunctional SDH mRNA and protein are generally much more abundant than their LKR/SDH counterparts. This supports the previous hypothesis (Tang et al., 2000; Galili et al., 2001) that the monofunctional SDH functions mainly to enhance the flux of lysine catabolism.
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Materials and methods |
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Plasmid construction and plant transformation
Construction of the HA-tagged LKR/SDH gene and the generation of the transgenic 7.5-HA plants containing this gene was reported previously (Tang et al., 2000
).
pLKR-GUS was constructed by combining the respective forward oligonucleotide 5'-CTAGATGCACATTCAACTCG-3' with reverse oligonucleotide 5'-AACTGTTTCAGCTAGAATCC-3' to amplify the specific 2143 bp genomic fragment including the first intron in the 5' non-coding region of gAtLKR/SDH and 72 bp of the LKR coding sequence. pSDH-GUS was constructed by combining the respective forward oligonucleotide 5'-TAAGCGTATCCATCATGATGCCTTGT-3' with reverse oligonucleotide 5'-TTCAGGCTTCTCTTTTATCTC-3' to amplify the specific 2834 bp genomic fragment of gAtLKR/SDH. These sequences were subcloned into an expression cassette containing the GUS coding sequence, followed by a nopaline synthase terminator of Agrobacterium tumefaciens and then into pPZP111 (Hajdukiewicz et al., 1994) to generate pPZP111-pLKR-GUS and pPZP111-pSDH-GUS.
Transformation of whole Arabidopsis plants was performed as described previously (Clough and Bent, 1998).
Plant material, growth conditions, and treatments
Wild-type (C-24 ecotype) and transgenic Arabidopsis thaliana seeds were germinated in a growth chamber under a 16 h light/8 h dark cycle on Nitch plates, containing Nitch complete medium, pH 5.8 (Duchefa, Haarlem, The Netherlands) supplemented with 2% sucrose and 0.8% plant agar (Difco, Detroit, MI). Fifty micrograms of kanamycin was added as a selection for the growth medium of the transgenic plants. Six-day-old seedlings were transferred to the liquid B5 medium consisting of Gamborg B5 complete medium (Duchefa) supplemented with 2% sucrose and 20 mM MES, pH 6, and incubated by shaking at 120 rpm for 4 d. On the ninth day after culturing, seedlings were subjected to the different hormone or metabolic treatments, as described in the figure legends. Abscisic acid (ABA), methyl jasmonate (MeJa), lysine, and NaCl were added at the desired concentrations. Seedlings were then incubated for an additional 24 h, harvested, and immediately frozen in liquid nitrogen. For carbon and nitrogen starvation treatments, 9-d-old seedlings were thoroughly rinsed with sterile double-distilled water, transferred to fresh sucrose or nitrogen-free medium and incubated for 72 h. In the nitrogen-free medium, ammonium sulphate and potassium nitrate were omitted from the standard B5 medium and NaH2PO4 was replaced with KH2PO4. For dehydration treatments, wild-type and transgenic Arabidopsis seedlings were grown for 12 d on MS medium containing Murashige and Skoog salts, 3% sucrose, and 0.8% plant agar, and removed from the agar to plastic dishes at 22 °C under dim light for various periods.
Nine-day-old transgenic Arabidopsis seedlings expressing the GUS gene grown under the conditions of the study were treated with 50 µM ABA and 50 µM MeJa for 24 h. For sugar starvation treatment, water-washed transgenic seedlings containing promoter–GUS constructs were transferred to the fresh medium with no sucrose, or supplemented with 2% glucose, 2% fructose, or 2% mannitol, and incubated for an additional 24 h before harvesting for GUS activity assay and for 72 h for histochemical staining.
For amino acid analysis, 9-d-old wild-type (ecotype WS) and homozygous LKR/SDH knockout seedlings were exposed to either 10 µM ABA for 24 h or to sugar starvation for 72 h, as described above.
Stock solutions of ABA and MeJa were prepared in ethanol and added to the media aseptically at desired concentrations. In the control flasks of related treatments, equal volumes of ethanol were added.
Northern blots, SDS-PAGE, and western blot analyses
Northern blots, SDS-PAGE, and western blot analyses were performed as previously described (Stepansky and Galili, 2003). For a probe to the northern blot, the SDH region of a full-length AtLKR/SDH cDNA was amplified by PCR (oligonucleotide primers 5'-ATGACGAAAAAATCAGGTGTT-3' and 5'-TATCATTCTGCCTCCTCCATCAG-3').
Assays of GUS activity and histochemical staining
Fluorometric analysis of GUS activity was carried as described previously (Jefferson et al., 1987). GUS activity was expressed as picomoles of 4-methylumbelliferone (4-MU) per milligram of protein per minute.
Histochemical GUS staining was performed on whole seedlings that were transferred to 15 ml falcon tubes and incubated at 37 °C for 16–18 h in GUS-staining solution containing 100 mM sodium phosphate buffer (pH 7.0), 1 mM EDTA, 1% Triton X-100, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 0.5 mg ml–1 X-Gluc. The staining solution was then removed and replaced with several changes of 70% ethanol until chlorophyll pigments were completely bleached.
Amino acid analysis
Free amino acids were extracted from whole seedlings as previously described (Karchi et al., 1993). Amino acid analysis was performed by reverse-phase HPLC on a C-18 column (Waters, Milford, MA) using precolumn phenylisothiocyanate derivatization.
Computerized promoter analysis
Prediction of putative regulatory elements was obtained by the following Web programs http://oberon.rug.ac.be:8080/PlantCARE/index.html and http://www.dna.affrc.go.jp/htdocs/PLACE (Higo et al., 1999).
Statistical analysis
The GUS activity and amino acids analyses were statistically evaluated, using the FIT Model of JMP 5.0 (SAS Institute Inc. Cary, NC, USA).
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Results |
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Localization of putative ABA, MeJa, sugar starvation, high salinity, and drought-responsive elements in the DNA regions upstream of the LKR/SDH and monofunctional SDH coding regions of the AtLKR/SDH gene
It has been shown recently that production of the Arabidopsis bifunctional LKR/SDH mRNA and polypeptide is strongly regulated by the two hormones, ABA and MeJa, as well as by various metabolic signals, particularly sugar starvation (Stepansky and Galili, 2003
). To test further how these hormones and metabolic signals regulate the expression of both the bifunctional LKR/SDH and monofunctional SDH gene products, an analysis was done to find out if the DNA regions upstream of the LKR/SDH and monofunctional SDH coding sequences of the AtLKR/SDH gene contain any putative ABA, jasmonate, and sugar-starvation responsive elements. As shown in Fig. 1, several putative consensus ABA responsive and sugar-starvation responsive elements were present in both DNA regions, but consensus jasmonate responsive elements were found only in the DNA region upstream of the LKR/SDH coding region. Putative consensus drought and high salinity responsive elements were present only in the DNA region upstream of the monofunctional SDH coding sequence.
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To test whether the presence of putative regulatory elements described in Fig. 1 is associated with concerted regulation of expression of the bifunctional LKR/SDH and monofunctional SDH products of the AtLKR/SDH gene, two different Arabidopsis lines were used. Wild-type Arabidopsis lines were used to detect the LKR/SDH and monofunctional SDH mRNAs, using a probe derived from the SDH coding region of a full-length LKR/SDH cDNA. However, it was not possible to use wild-type Arabidopsis to study the production of the monofunctional SDH polypeptides because there are no antibodies against the SDH polypeptide. Thus, for protein analysis, a previously reported transgenic Arabidopsis genotype (Tang et al., 2000
), which expresses the Arabidopsis LKR/SDH gene (with its own promoter and coding region including exons and introns) to which an HA epitope tag was fused in-frame at the end of the SDH coding region, was used. This genotype encodes both bifunctional LKR/SDH and monofunctional SDH polypeptides containing C-terminal HA epitopes and hence allows their detection with commercial anti-HA monoclonal antibodies. Several independently transformed lines of this genotype were initially tested with comparable results; therefore, the results of only one of these lines (line 7.5-HA-1; Tang et al., 2000) will be presented.
Effect of ABA and MeJa on the production of LKR/SDH and monofunctional SDH mRNAs and polypeptides
To test the effects of ABA and MeJa on the concerted production of the bifunctional LKR/SDH and monofunctional SDH mRNAs and polypeptides from the composite AtLKR/SDH locus, young shoots of wild-type Arabidopsis and the transgenic line 7.5-HA-1, grown in liquid media, were exposed to different concentrations of ABA and MeJa for 24 h. As shown in Figs 2 and 3 (lanes a), in control non-treated plants the levels of the bifunctional LKR/SDH mRNA and polypeptide were significantly lower than those of the monofunctional SDH mRNA and polypeptide. Treatment with ABA or MeJa stimulated the levels of both LKR/SDH and monofunctional SDH mRNAs, but the degree of stimulation (compared with control non-treated plants) was significantly higher for the LKR/SDH mRNA (Figs 2A and 3A; compare lane a with lanes b–d). A similar stimulation was also observed for the bifunctional LKR/SDH polypeptide, while none of these treatments noticeably affected the levels of the monofunctional SDH polypeptide that was significantly more abundant than the bifunctional LKR/SDH polypeptide on the immunoblots (Figs 2B and 3B; compare lane a with lanes b–d). To gain a more accurate quantitative estimation of the monofunctional SDH polypeptide following the different ABA and MeJa treatments, it was also analysed after a serious of protein dilutions. These dilutions showed that all concentrations of MeJa had no significant effect on the level of the monofunctional SDH polypeptide, while the highest concentration of ABA (50 µM) caused a small 
2-fold increase in the level of this polypeptide compared with the control non-treated plants (data not shown).
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Effects of carbon and nitrogen status on the production of the LKR/SDH and monofunctional SDH mRNAs and polypeptides
At the next stage, the effect of sugar and nitrogen starvation on the production of the LKR/SDH and monofunctional SDH mRNAs and polypeptides was tested. This was done by growing cultures of the wild-type and 7.5-HA-1 plants for 9 d in a medium containing 2% sucrose (control sugar level) and then transferring them to new media containing 2% or 0% sucrose (sugar starvation) for an additional 3 d, or 5% sucrose (excess sugar) for an additional 24 h (Fig. 4, lanes a–c). The levels of the bifunctional LKR/SDH and monofunctional SDH mRNAs were enhanced upon exposure to sugar starvation (with a more pronounced degree of stimulation for the bifunctional LKR/SDH mRNA), while 5% sucrose had no significant effect on their levels (Fig. 4A, compare lane a with lanes b and c). A similar stimulatory response to sugar starvation was also observed for the bifunctional LKR/SDH polypeptide, while the level of the more abundant monofunctional SDH polypeptide was not changed significantly (Fig. 4B; compare lane a with lanes b and c).
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Next, it was tested whether the effect of sugar starvation can be modulated by the addition of sucrose or other sugars to sugar-starved cultures. The stimulation of production of the bifunctional LKR/SDH and monofunctional SDH mRNAs by sugar starvation was reversed by the addition of sucrose, glucose, and fructose, but not mannitol, indicating that the sugar effect was not specific to sucrose and was not related to a change in osmotic conditions (Fig. 5A; compare lane a with lanes b–f). The changes in the level of the bifunctional LKR/SDH polypeptide paralleled those observed on the mRNA level, while the level of the more abundant monofunctional SDH polypeptide was again not changed significantly (Fig. 5B, compare lane a with lanes b–f). It also seems that both RNA and protein levels of LKR/SDH increased slightly in response to mannitol. This induction of LKR/SDH is apparently due to its stimulation by endogenous ABA levels, which are increased in plants in response to mannitol (Han et al., 2004
).Next to be tested was the effect of nitrogen starvation, either alone or in combination with sugar starvation, on the relative levels of the LKR/SDH and monofunctional SDH mRNAs and polypeptides. To this end, 9-d-old cultures of wild-type and 7.5-HA-1 plants were transferred to new media lacking nitrogen for an additional 3 d, or to new media lacking both nitrogen and sugars for 24 h after adaptation in sugar-free media for 2 d. As shown in Fig. 6A (lanes a–c), in contrast to the stimulatory effect of sugar starvation, exposure of the Arabidopsis cultures to nitrogen starvation slightly reduced the levels of both bifunctional LKR/SDH and monofunctional SDH mRNAs. Exposure of the Arabidopsis cultures to 3 d in medium lacking both nitrogen and sugars caused some stimulation of the LKR/SDH mRNAs, whose level was in-between those observed in sugar-starved or nitrogen-starved cultures (Fig. 6A, lanes b–d). The changes in the level of the bifunctional LKR/SDH polypeptide paralleled in general those observed on the mRNA level (Fig. 6B, lanes a–d), although the level of this polypeptide was appreciably repressed in nitrogen-starved compared with control non-treated plants (Fig. 6B, lane c). Again the level of the more abundant monofunctional SDH polypeptide was not significantly changed in response to sugar and nitrogen starvation (Fig. 6B, lanes a–d).
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The effect of lysine on the levels of LKR/SDH and monofunctional SDH mRNAs and polypeptides was also tested. As shown in Fig. 7A, lysine stimulated the levels of both LKR/SDH and monofunctional SDH mRNAs in wild-type seedlings. A similar stimulatory response to lysine was also observed for the bifunctional LKR/SDH polypeptide, whereas the level of the more abundant monofunctional SDH polypeptide was not significantly changed (Fig. 7B).
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Transcriptional regulation of the production of the LKR/SDH and monofunctional SDH mRNAs by hormones and metabolic signals
Next, it was desirable to find out whether the hormonal and metabolic effects on the production of the two AtLKR/SDH gene products are regulated at the transcriptional level and whether both the upstream and internal promoter regions were responsible to these hormones. This was addressed by transforming Arabidopsis plants with two GUS reporter constructs, in which the coding sequence of GUS was fused to DNA sequences of 2073 bp and 2834 bp, located upstream of the LKR/SDH (pLKR-GUS) and monofunctional SDH (pSDH-GUS) coding regions, respectively. A number of independently transformed lines were selected for each of these constructs. Most of the independently transformed lines for each of these constructs showed very similar patterns of GUS expression in response to the various metabolic and hormonal signals, and therefore detailed studies were performed on six representative lines of each of these two transgenic genotypes.
Analyses of the effects of ABA, MeJa, and sugars on GUS activities in these transgenic plants were performed in cultured plants grown in an identical manner to the plants used for the experiments described in Figs 2–5
. Exposure of the pLKR-GUS-expressing plants to ABA and MeJa caused an average of 5- and 2.5-fold increases in GUS activity, respectively (Fig. 8A). In contrast, GUS activity in the pSDH-GUS plants was stimulated on average only 2.5-fold by ABA and was not significantly stimulated by MeJa (Fig. 8B). As shown in Fig. 9A, GUS activity in the pLKR-GUS-expressing plants was also stimulated on average 3-fold by sugar starvation, and this stimulation was not repressed by feeding with mannitol as a sole sugar. Feeding with fructose or glucose as the sole sugars slightly reduced the induction rate obtained by sugar starvation, causing only 2-fold stimulation (Fig. 9A). GUS activity in the pSDH-GUS-expressing plants exhibited, on average, a smaller 1.5-fold stimulation by sugar starvation, while no significant stimulation was observed upon feeding the plants with mannitol, fructose, and glucose as sole sugars (Fig. 9B).
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In situ GUS expression pattern in roots and shoots of plants expressing the pLKR-GUS and pSDH-GUS constructs
It was also of interest to test whether the DNAs upstream of the AtLKR/SDH and monofunctional AtSDH coding regions regulate similar or distinct in situ expression patterns under regular growth conditions and in response to hormonal and metabolic signals. In regularly grown plants, the pLKR-GUS construct was expressed in young leaves of cotyledons as well as in root tips and mature root parts, but not in the region containing the elongation zone (Fig. 10,A1–A3). The pSDH-GUS was most profoundly expressed in the cotyledons, and to a very low level in all root parts (Fig. 11, A1–A3). Exposure of the pLKR-GUS-expressing plants to ABA significantly stimulated GUS production in the cotyledons and young leaves as well as in all root parts including the elongation zone (Fig. 10, B1–B3). Exposure to ABA had a small positive effect on GUS staining in leaves and roots of plants expressing the pSDH-GUS construct, while a significant stimulation of GUS expression was observed in the root elongation zone (Fig. 11, B1–B3). Exposure of the pLKR-GUS-expressing plants to MeJa and sugar starvation significantly stimulated GUS production in the cotyledons, leaves, and roots (Fig. 10, C1–C3, D1–D3 ), but the stimulation of GUS production in the root elongation zones was significantly less pronounced than upon exposure to ABA (Fig. 10; compare B3 with C3 and D3). Exposure to MeJa and sugar starvation had only small positive effects on GUS staining in roots and shoots of plants expressing the pSDH-GUS construct (Fig. 11, C1–C3, D1–D3).
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Effect of salt and drought treatments on the production of the LKR/SDH and monofunctional SDH mRNAs and polypeptides
Previous reports (Deleu et al., 1999
; Moulin et al., 2000) showed that production of both the bifunctional LKR/SDH and monofunctional SDH gene products is stimulated upon exposure of rape seed plants to PEG. To find out if expression of the AtLKR/SDH gene is affected by salt and drought stresses the following test was done. To impose salt stress, cultures of wild-type and 7.5-HA-1 plants were grown for 9 d in regular media (see Materials and methods) and then transferred to new media containing increasing concentrations of NaCl for 24 h. As shown in Fig. 12, treatment of NaCl caused only a small induction in the levels of the LKR/SDH and non-functional SDH mRNAs and polypeptides, which was evident mostly in the highest concentrations of 120 mM NaCl, which is known to cause severe salt stress to plants. To impose drought stress, wild-type and 7.5-HA-1 plants were grown on agar-plates for 12 d and then removed to plastic dishes with open lids (air drying) for various periods. As shown in Fig. 13, the drought stresses only caused a small increase in the levels of the LKR/SDH and non-functional SDH mRNAs and polypeptides.
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Stimulation of the LKR/SDH gene expression upon exposure to ABA and sugar starvation is associated with enhanced lysine catabolism
Since expression of the LKR/SDH gene was significantly stimulated by various hormonal and metabolic signals, it was also desirable to evaluate whether this stimulation is correlated with enhanced lysine catabolism, using ABA and sugar-starvation treatments as examples. This was addressed using a homozygous Arabidopsis LKR/SDH knockout mutant that lacks lysine catabolism (Zhu et al., 2001
). Cultured wild-type and homozygous LKR/SDH knockout seedlings were exposed to either ABA or sugar starvation (see Materials and methods) and the relative free lysine levels were analysed. As shown in Fig. 14, in non-treated plants (control) both lines accumulated comparable, relatively low, levels of free lysine. Exposure to either ABA or sugar starvation stimulated the free lysine levels in the wild-type seedlings, although sugar starvation had a much stronger effect than ABA. Notably, free lysine levels in the LKR/SDH knockout seedlings exposed to either ABA or sugar starvation were significantly higher than in the wild-type seedlings exposed to the same treatment.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Rationale of the present research and suitability of the experimental approach
It has recently been shown that production of the bifunctional LKR/SDH mRNA and polypeptide in Arabidopsis is regulated in concert by the hormones ABA and MeJa as well as by the status of sugars and nitrogen (Stepansky and Galili, 2003
). Yet, the Arabidopsis AtLKR/SDH locus also encodes an additional monofunctional SDH polypeptide that is transcribed from an internal promoter within the LKR/SDH coding region (Tang et al., 2000; Galili et al., 2001), the function of which is still unclear. Aiming at elucidating the regulatory function of this enzyme in lysine catabolism, the hormonal and metabolic controls of production of the monofunctional SDH mRNA and protein, relative to that of its LKR/SDH counterpart, have now been examined. While the levels of the LKR/SDH and monofunctional SDH mRNAs could be analysed in wild-type plants using a probe derived from the SDH domain of the AtLKR/SDH open reading frame, the levels of their corresponding polypeptides were analysed in transgenic 7.5-HA plants (Tang et al., 2000). These plants express an AtLKR/SDH gene (with its own promoter and the entire set of exons and introns) with an HA epitope tag fused in-frame upstream of the stop codon of the LKR/SDH open reading frame, enabling the detection of both the LKR/SDH and monofunctional SDH polypeptides by commercial anti-HA monoclonal antibodies. The levels of the bifunctional LKR/SDH polypeptide in the 7.5-HA plants (this paper) and in wild-type plants (Stepansky and Galili, 2003; LKR/SDH, but not SDH, was specifically detected by anti-LKR monoclonal antibodies) were nearly identically affected by the various hormonal and metabolic treatments, confirming the suitability of 7.5-HA for this analysis.
The levels of the monofunctional SDH mRNA and polypeptide are more abundant and less responsive to hormonal and metabolic stimuli than their bifunctional LKR/SDH counterparts
In non-treated plants, the levels of both monofunctional SDH mRNA and protein were significantly higher than their bifunctional LKR/SDH counterparts. Treatment with ABA, but not MeJa, as well as exposure to the various sugar and nitrogen regimes, affected the level of the LKR/SDH and monofunctional SDH mRNAs in the same way but, in most cases, the level of the monofunctional SDH mRNA was significantly less responsive to these treatments (Figs 2–7, 12, and 13). Yet, the level of the monofunctional SDH mRNA remained higher or became roughly equal to that of its LKR/SDH counterpart in all treatments. The level of the monofunctional SDH polypeptide was essentially not affected by any of the treatments and also remained either higher or became roughly equal to that of its LKR/SDH counterpart in all treatments. This implies that the major regulation of lysine catabolism by the hormonal and metabolic signals is exerted by LKR rather than by SDH, while the level of SDH enzyme, although slightly modulated, remains high to enable efficient flux of lysine catabolism. The high total level of the SDH relative to the LKR enzymes apparently compensates for the pH optima of 9 for SDH activity, which is significantly higher than the natural pH of the cytosol where this enzyme resides (Galili et al., 2001).
Stimulation of LKR/SDH gene expression by lysine
The present results showed that lysine stimulated the production of the LKR/SDH mRNA and protein levels and, to some extent, the monofunctional SDH mRNA as well, but not protein. This supports the previous observation in which lysine overproduction in developing tobacco seeds caused a 10-fold stimulation of LKR activity and that this stimulation was dependent on calcium and protein phosphorylation/dephosphorylation (Karchi et al., 1994, 1995). Taken together, these results imply that plant cells can sense the level of lysine and stimulate lysine catabolism via a number of regulatory processes.
The differential production of the bifunctional LKR/SDH and monofunctional SDH polypeptides in response to hormonal and metabolic signals is regulated at the transcriptional level
The contribution of transcriptional control to the differential accumulation of the LKR/SDH and monofunctional SDH gene products was also tested, using GUS constructs containing the promoters upstream of the LKR/SDH and monofunctional SDH coding regions (Figs 8, 9). Quantitative analysis of GUS activity in extracts from whole seedlings grown in culture showed that the promoter upstream of the LKR/SDH coding region was responsive, to various extents, to ABA, MeJa, and sugar treatments. In contrast, the internal promoter, upstream of the SDH coding region was induced only by ABA and the extent of its induction was smaller than that observed with the promoter upstream of the LKR/SDH coding region. These results show that the differential hormonal and metabolic effects on the production of the LKR/SDH and monofunctional SDH polypeptides are largely (although not necessarily entirely) regulated at the transcriptional level. These results are also in agreement with the DNA sequence analysis of the two promoters. While both the LKR/SDH and SDH promoters contain a number of putative ABA responsive elements (ABRE), the LKR/SDH promoter also contains a number of putative sugar-starvation-responsive elements, compared with only one such putative element in the SDH promoter (Fig. 1). In addition, the LKR/SDH, but not the SDH promoter, contains MeJa responsive elements (Fig. 1).
The reason for the significantly more abundant levels of monofunctional SDH than bifunctional LKR/SDH polypeptides in all of the western blots is not clear. Notably, in contrast to this, the pLKR-GUS- and pSDH-GUS-expressing plants contained comparable GUS activities (Figs 8, 9). It is therefore hypothesized that the higher level of the monofunctional SDH than the bifunctional LKR/SDH polypeptides is also due to post-transcriptional control. Yet, at present, the possibility cannot be ruled out that transcription of the monofunctional SDH mRNA is also regulated by elements that are located in the promoter upstream of the LKR/SDH coding region, which are not present in the pSDH-GUS construct. However, such potential elements are also expected to increase the transcription of the LKR/SDH mRNA.
The LKR/SDH and monofunctional SDH promoters show distinct in situ expression patterns in roots and shoots
The present results, using the pLKR-GUS and pSDH-GUS transgenic plants, showed that these two promoters possess distinct in situ expression patterns and responses to hormonal and metabolic signals in roots and shoots of young Arabidopsis seedling. Under favourable growth conditions (no exposure to stress or hormones), the LKR/SDH promoter was expressed in the cotyledons and leaves, but its expression was much more abundant in root caps and mature roots, but not in the root elongation zone. By contrast, the SDH promoter was abundantly expressed in the cotyledons, but its expression was weaker in the roots and leaves compared with the LKR/SDH promoter. Although an in trans contribution of the LKR/SDH promoter to the transcription of the monofunctional SDH mRNA cannot be ruled out, these results suggest that, under favourable growth conditions, the cotyledons require more extensive lysine catabolism (both LKR/SDH and monofunctional SDH), than roots. The specific in situ expression pattern of the LKR/SDH gene in roots and shoots of plants grown under favourable growth conditions is also in accordance with the expected function of the lysine catabolism pathway during plant development. The cotyledons as well as the mature root zones are source tissues, which remobilize metabolites into actively growing young leaves. The cotyledons remobilize storage compounds that are produced during seed development, while mature roots remobilize nitrogenous compounds (like amino acids) that are produced in actively growing root zones (elongation zone) via nitrogen assimilation into glutamine and glutamate. These remobilization processes require the activation of catabolic pathways, like the pathway of lysine catabolism.
Since nitrogen assimilation and the build-up of amino acids takes place in the actively growing parts of the roots, particularly the elongation zones, catabolic pathways are expected to be repressed in these tissues. Indeed, the present results showed that, under favourable growth conditions expression of the LKR/SDH gene is strongly repressed in the root elongation zone.
The strong expression of the LKR/SDH gene in root caps is also expected based on the function of this region to enable root penetration into the soil. Penetration into the soil is associated with extensive cell death due to mechanical forces and formation of new cells. High activity of catabolic pathways in this region may enable the remobilization of metabolites from the old to the new cells.
Notably, although the LKR/SDH and monofunctional SDH promoters are not abundantly expressed in root elongation zones and the young true leaves, exposure to ABA significantly stimulated the expression of these two promoters in these tissues and also increased their expression in the cotyledons and other root tissues. This implies that ABA overrides other potential repressors that repress the expression of the AtLKR/SDH gene in the root elongation zone. The physiological significance of this effect has yet to be elucidated. The stimulation of expression of both the LKR/SDH and SDH promoters of the AtLKR/SDH gene by ABA also suggests that these two promoters are regulated in a concerted manner by a number of regulatory factors that either repress or activate the production of the LKR/SDH and monofunctional SDH gene products.
By contrast to ABA, MeJa did not stimulate the expression of the LKR/SDH gene in root elongation zones and in the young true leaves. Yet MeJa further stimulated the expression of this gene in tissues where the gene is also abundantly expressed in regularly grown plants. MeJa is involved in a number of functions during plant growth, including the response to various biotic and abiotic stresses as well as senescence. The present results therefore suggest that MeJa regulates lysine catabolism, particularly in senescing tissues, and is therefore mainly involved in the function of this pathway in metabolite remobilization.
Regulatory role of lysine catabolism under drought and salt stresses
Since one of the major products of Lys catabolism is glutamate, which is a precursor for many stress-associated metabolites (Galili et al., 2001), it was interesting to elucidate whether this pathway also participates in the metabolic response of plants to abiotic stresses. The participation of Lys catabolism in osmotic stress was suggested by the fact that expression of the LKR/SDH gene of rape seed is induced upon exposure to PEG (Deleu et al., 1999; Moulin et al., 2000). Under the present experimental conditions, expression of the AtLKR/SDH gene was only slightly induced by drought and salt stresses and also only under severe stress conditions. This is in agreement with only one each of putative salt and drought responsive elements being identified in the internal SDH promoter with none being identified in the LKR/SDH promoter (Fig. 1). Hence, although it is impossible to generate a general conclusion from the present experiments, they suggest that Lysine catabolism may not participate extensively in stress-associated metabolism under all physiological and developmental programmes.
Functional significance of Lys metabolism in the response of plants to hormonal and metabolic signals
In wild-type plants grown under favourable growth conditions, lysine generally accumulates to relatively low levels due to the sensitivity of dihydrodipicolinate synthase to feedback inhibition by lysine (Galili, 1995). Yet, various hormonal, metabolic, and stress signals can trigger the degradation of cellular proteins, resulting in significant accumulation of free lysine, which should be further catabolized by enhanced expression of the LKR/SDH gene. In the presented report, this hypothesis was also partially tested by a functional approach, using an Arabidopsis LKR/SDH knockout mutant that lacks lysine catabolism (Zhu et al., 2001). The present results showed that exposure of plants to ABA and sugar starvation, as examples of hormonal and metabolic signals, increase free lysine levels in wild-type plants, although to a different extent. Moreover, upon exposure to ABA and sugar starvation, free lysine levels were significantly higher in the LKR/SDH knockout mutant than in the wild type, implying that stimulation of LKR/SDH gene expression by these treatments is associated with a significant increase in lysine catabolism.
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Acknowledgements |
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This work was supported by The United States–Israel Binational Agricultural Research and Development (BARD) Fund, grant no. IS-333102. GG is an incumbent of the Bronfman Chair in Plant Sciences.
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