JXB Advance Access originally published online on February 27, 2004
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Journal of Experimental Botany, Vol. 55, No. 398, pp. 791-801, April 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
The auxin conjugate 1-O-indole-3-acetyl-ß-D-glucose is synthesized in immature legume seeds by IAGlc synthase and may be used for modification of some high molecular weight compounds
Received 17 July 2003; Accepted 16 December 2003
Nicholas Copernicus University, Institute of General and Molecular Biology, Department of Biochemistry, ul. Gagarina 9, 87-100 Toruñ, Poland
* To whom correspondence should be addressed. Fax: +48 56 611 44 72. E-mail: anjakubo{at}cc.uni.torun.pl
Abbreviations: IAA, indole-3-acetic acid; 1-O-IAGlc, 1-O-(indole-3-acetyl)-ß-D-glucose; IAInos, indole-3-acetyl-myo-inositol; IAGlc synthase, UDP-glucose:indole-3-acetate glucosyltransferase; IAInos synthase, 1-O-(indole-3-acetyl)-glucose:myo-inositol indoleacetyl transferase.
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Immature seeds of some dicotyledonous plants contain IAGlc synthase catalysing the synthesis of 1-O-IAGlc. This enzyme activity is comparable with 1-O-IAGlc synthase activity investigated earlier in liquid endosperm of Zea mays. Polyclonal antibodies against maize 1-O-IAGlc synthase cross-react with partially purified 1-O-IAGlc synthase from immature pea and rape seeds. Single immunoreactive bands were observed at a locus corresponding to 45.7 kDa and 43.7 kDa from pea and rape enzyme preparations, respectively, unlike that from the 50 kDa molecular mass of the maize enzyme. It was also observed that some high molecular weight compounds of pea seeds are labelled in vivo by [14C] IAA, and unlabelled 1-O-IAGlc inhibits that labelling. In immature pea seeds 43–49.8% of the IAA-modified high molecular weight compounds, obtained after ultracentrifugation, was found in the soluble fraction and 50.1–57% in the insoluble fraction. Ester-linked IAA accounted for about 6–9% and 38–45.6% in soluble and insoluble material, respectively, estimated after hydrolysis in 1 N NaOH. Enzymatic hydrolysis of IAA-labelled high molecular weight compounds gives free IAA and compound(s) corresponding to IAGlc isomers. These results suggest that 1-O-IAGlc synthesized in legume seeds may be used for the modification of some high molecular weight compounds.
Key words: High molecular weight indole-3-acetic acid conjugates, IAA-glucose synthase, indole-3-acetic acid, indole-3-acetic acid glucose, legume plants.
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Introduction |
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Indole-3-acetic acid (IAA) is the most abundant naturally occurring auxin involved in the control of plant growth and development. It is well established that IAA can occur either as the hormonally active free acid or in bound forms in which the carboxyl group is conjugated to sugars and myo-inositol via ester linkages or to amino acids or peptides via amide linkages (Cohen and Bandurski, 1982; Bandurski et al., 1995; Normanly, 1997; Slovin et al., 1999; Bartel et al., 2001; Ljung et al., 2002). IAA conjugates have been identified in a number of plant species, ranging from liverworts to angiosperms (Sztein et al., 1995, 2000). A limited number of species have been studied, including maize, Arabidopsis, tomato, bean, soybean, Scots pine, rice, oat, chestnut, and poplar, although the most extensively studied have been seeds of maize, bean, and soybean (Slovin et al., 1999). Maize kernels contain primarily ester-linked conjugates, including IAA-glucose, IAA-myo-inositol, IAA-myo-inositol glycosides, and a large cellulosic glucan conjugate that together represent about 97–99% of the total IAA in the seed endosperm (Bandurski and Schulze, 1977; Cohen and Bandurski, 1982; Bandurski et al., 1995; Slovin et al., 1999). Esterified IAA is also the predominant conjugate in seeds of rice which contain approximately 62–70% ester-linked IAA (Bandurski and Schulze, 1977; Hall, 1980) and in the liquid endosperm of horse chestnut (Domagalski et al., 1987). Moreover, Percival and Bandurski (1976) described an IAA ester glucoprotein fraction, isolated from oat seeds, that accounted for about 80% of the IAA found in this tissue. In the early stages of bean seed development ester-linked IAA represents approximately 35%, and free IAA about 40%, of the total IAA pool (Bialek and Cohen, 1989). The level of esterified and free IAA declines rapidly during seed maturation, so that in fully mature seeds the ester-linked IAA represents about 13% of the total IAA pool, and only 6% is free IAA. It is noteworthy, that in seeds harvested at full maturity, IAA is conjugated to several polypeptides and proteins that approximate 80% of the total IAA pool (Bialek and Cohen, 1986, 1989; Walz et al., 2002). Soybean seeds have become another dicotyledonous plant material in which essentially all small molecular mass IAA conjugates have been identified and assayed. Quantitative evaluation indicates that conjugates with aspartate and glutamate are the predominant IAA constituents present in these seeds (Cohen, 1982; Epstein et al., 1986).
The entire complement of IAA conjugates has also been determined in vegetative tissues of several plant species. It was shown that ester conjugates of IAA constitute up to 80% of the IAA in the shoots of young dark-grown maize, and IA-myo-inositol represents about 19% of these compounds (Pengelly et al., 1982; Chisnell, 1984). In tomato pericarp, IAA undergoes conjugation to yield both IAA-glucose and amide-linked IAA, and the preferential formation of either IAA-glucose or amide-linked IAA conjugates depends on the ripening stage of the fruit (Catalá et al., 1992; Iyer et al., 1997). Henbane cells, tobacco explants, and tobacco leaf protoplasts produce mainly auxin-aspartate and auxin-glucose conjugates (IAA- or NAA-conjugates) (Caboche et al., 1984; Delbarre et al., 1994; Oetiker and Aeschbacher, 1997; Smulders et al., 1990). Arabidopsis, the model dicotyledonous plant, is also able to form the ester-linked IAA conjugates that constitute approximately 8–10% of the total IAA pool (Tam et al., 2000). In addition, analyses have shown the presence of IA-alanine, IA-leucine, IA-aspartate, IA-glutamate, and IA-glutamine, although, in total, the small molecular mass conjugates together make up only about 2–3% of the conjugate pool in the whole plant (Östin et al., 1998; Barratt et al., 1999; Barlier et al., 2000; Kowalczyk and Sandberg, 2001; Tam et al., 2000). Because amide-linked IAA conjugates constitute approximately 90% of the IAA pool, the high molecular weight covalent complexes, apparently IAA-proteins, may represent the major form of auxin in Arabidopsis, and in other dicotyledonous plants as well.
Despite efforts from several laboratories there has been no success in purifying the enzyme from plant tissues catalysing the synthesis of low or high molecular weight amide-linked conjugates. The enzyme responsible for the synthesis of IAA-
-L-lysine in the plant pathogen Pseudomonas savastanoi was studied and the gene for this enzyme has been cloned (Glass and Kosuge, 1986; Roberto et al., 1990). Recently, Staswick et al. (2002) suggested that IAA-amino acid conjugates can be produced via an adenylation intermediate. On the other hand, a comparatively large amount of information is now available on the biosynthesis of the IAA-ester conjugates in immature kernels of Zea mays. Studies by Michalczuk and Bandurski (1980, 1982) indicated that the formation of the acyl alkyl acetal 1-O-(indole-3-acetyl)-ß-D-glucose (IAGlc) by indole-3-acetylglucose synthase (reaction 1) is the first step in the series of reactions leading to the IAA–ester conjugates found in maize:
IAA+UDP-glucose
1-O-IA-glucose+UDP (reaction 1)
UDP-glucose:IAA glucosyltransferase catalysing this reaction has been extensively purified from corn endosperm and polyclonal antibodies have been produced (Kowalczyk and Bandurski, 1991; Leznicki and Bandurski, 1988). Szerszen et al. (1994) have used antibodies to select a cDNA clone for the IAGlc synthase from a maize library. Recently, an Arabidopsis gene encoding UDP-glucosyltransferase that forms 1-O-IA-glucose was identified (Jackson et al., 2001). Moreover, the stimulation of IAGlc synthase gene expression by auxin in maize coleoptiles has been observed (Kowalczyk et al., 2002).
In immature kernels of Zea mays the energetically unfavourable synthesis of 1-O-IA-glucose is followed by an energetically favorable transacylation of the IAA moiety from 1-O-IA-glucose to myo-inositol (reaction 2):
1-O-IA-glucose+myo-inositolIA-myo-inositol+glucose (reaction 2)
The synthesis of IA-myo-inositol (IAInos) was observed for the first time in vitro in the seeds of maize (Michalczuk and Bandurski, 1980, 1982), and the transferase catalysing this reaction was partially purified and characterized (Kesy and Bandurski, 1990). Recently, a six-step procedure was described for the purification of an electrophoretically homogenous IAInos synthase displaying similarity to the serine carboxypeptidase-like acyltransferases family (Kowalczyk et al., 2003).
In this report, it is indicated for the first time that 1-O-IAGlc synthase activity is present in immature seeds of some dicotyledonous plants. Partially purified 1-O-IAGlc synthase from immature pea and rape seeds cross-reacts with polyclonal antibodies against maize 1-O-IAGlc. It is also observed that some high molecular compounds of pea seeds are labelled in vivo by [14C] IAA.
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Materials and methods |
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Plant material
Plants used for analysis were grown under field conditions, during the summers of 2002 and 2003. The young pods with immature seeds were harvested and the seeds were selected according to the length of their long axis. The pods of pea (Pisum sativum) were harvested at different times of seed development until full seed maturity. For enzyme purification the immature seeds were frozen immediately after harvest and were stored in freezer bags at –20 °C.
Chemicals
Chemicals were obtained from Gibco (USA), ICN (USA), Merck (Germany), and Sigma (Germany). [2'-14C] IAA and D-[U-14C] glucose were from Amersham (UK). 1-O-IAGlc was synthesized by Dr Antoni Le
nicki using the methods of Jakas et al. (1993), IAA-myo-inositol was chemically synthesized according to the method of Nowacki et al. (1978). 1-O-IAGlc synthase and IAInos synthase preparations were purified from immature corn endosperm according to the methods of Kowalczyk and Bandurski (1991) and Kowalczyk et al. (2003).
Tissue homogenates for 1-O-IAGlc synthase activity assay
1 g of seeds or vegetative tissue from selected plants was homogenized at 0–4 °C in a glass homogenizer with 1 ml of 50 mM TRIS-HCl buffer, pH 7.6 containing 2 mM EDTA, 2 mM 2-mercaptoethanol, and PMSF (100 µg ml–1). The homogenates were centrifuged at 10 000 g for 10 min, and enzyme activity was assayed in the supernatant fluid.
Purification of 1-O-IAGlc synthase from immature pea seeds
A 200 g portion of frozen pea seeds was homogenized with 200 ml of 50 mM TRIS-HCl buffer, pH 7.6 containing 2 mM EDTA, 2 mM 2-mercaptoethanol, and PMSF (100 µg ml–1) using a mortar and pestle, and then a Polytron homogenizer. All purification steps were performed at 4 °C. The homogenate was centrifuged at 16 000 g for 15 min (step I). To the resultant supernatant, 45% (w/v) polyethylene glycol 6000 in 25 mM TRIS-HCl, pH 7.6, containing 2 mM EDTA and 2 mM 2-mercaptoethanol was added slowly, with stirring, to obtain a 15% (w/v) final concentration of polyethylene glycol. The solution was stirred in ice for 30 min, and after centrifugation for 60 min at 22 000 g a clear supernatant fluid was obtained (step II). The supernatant fluid was applied to a DEAE-Sephacel column (2.5x12 cm) equilibrated with 25 mM TRIS-HCl, pH 7.6 containing 1 mM 2-mercatoethanol and PMSF (50 µg ml–1). The column was washed with the same buffer until the A280 decreased to baseline. Bound proteins were eluted with 80 mM NaCl and then with 150 mM NaCl in the buffer. The fractions containing enzyme activity were pooled (step III). The combined fractions were diluted with 10 mM TRIS-HCl, pH 7.6 containing 1 mM 2-mercaptoethanol and PMSF (50 µg ml–1) and applied to a hydroxylapatite column (1x3 cm). The column was washed with equilibrating buffer (25 mM TRIS-HCl, pH 7.6, 1 mM 2-mercaptoethanol and PMSF 50 µg ml–1) and proteins were eluted with 1 mM phosphate (K) buffer, pH 7.4 containing 1 mM 2-mercaptoethanol and PMSF (50 µg ml–1), and then with 10 mM phosphate (K) buffer. The active fractions were pooled and concentrated by ultrafiltration using an Amicon Diaflo YM-10 filter (step IV). TSK-Gel Toyopearl DEAE-650M column (1x3 cm) was equilibrated with 25 mM TRIS-HCl buffer, pH 7.6 containing 1 mM 2-mercaptoethanol and PMSF (50 µg ml–1). The combined fractions were applied to the TSK-Gel column after a 5x dilution to decrease the phosphate concentration. The column was washed with the buffer, and the enzyme was eluted with a linear gradient of 0–150 mM NaCl with a total volume of 100 ml. Fractions containing IAGlc synthase activity were pooled and concentrated (step V).
Qualitative assay of IAA-amino acid, IAA-disaccharide, and IAA-glucose isomers synthesis
To examine formation of the IAA–amide conjugates or IAA-esters with disaccharides, fresh immature seeds were transferred to 25 ml Erlenmeyer flask containing 10 ml of 10 mM phosphate (K) buffer, pH 6.5, containing 25 µM IAA. The flask was shaken for 12 h at room temperature. The pretreated seeds were homogenized and synthesis of IAA conjugates was tested in a total volume of 8 µl containing 50 mM HEPES-NaOH buffer, pH 7.6, 1 mM 1-O-IAGlc, 2.5 mM MgCl2, and 5 mM amino acids (aspartate, glutamate or leucine) or 5 mM disaccharide (melibiose, sucrose, and maltose), or 1 mM glucose and 0.1 µCi of D-[U-14C] glucose (specific activity 293 mCi mmol–1), respectively. The reaction was started by the addition of 3 µl of homogenate and after a 2 h or 18 h incubation at 30 °C was stopped by drying a 4 µl aliquot on a Silica Gel 60 TLC plate. The reaction products were separated by thin layer chromatography (TLC) using as solvent ethyl acetate/methyl ethyl ketone/ethyl alcohol/water (5:3:1:1, by vol.) (Labarca et al., 1965). Indole compounds were detected by dipping the plate in the Ehmann reagent (Ehmann, 1977), blotting and drying for 5 min at 100 °C. The region corresponding to IAGlc isomers was scraped individually from the plate and placed into the vial with 4 ml of scintillation fluid EcoLite (ICN). The radioactivity was measured in a Wallac 1409 liquid scintillation counter.
Labelling high molecular weight compounds by [2'-14C] IAA
0.5–2 g of immature pea seeds (3–4 mm) were rinsed in distilled water and were then placed on a Petri dish filled with 1–3 ml 10 mM phosphate (K) buffer, pH 6.5 containing 2% (w/v) sucrose, 1 mM UDPG, 2.5 µCi of [2'-14C] IAA (specific activity 50 mCi mmol–1), and 25 µM IAA. In a parallel experiment, the incubation medium contained in addition 0.5 mM unlabelled 1-O-IAGlc. Seeds were incubated on a shaker at 25 °C in a dark room. After a 48 h incubation, the seeds were rinsed many times with distilled water and homogenized in 2–9 ml of 100 mM TRIS-HCl buffer, pH 7.6 containing 150 mM NaCl, 5 mM 2-mercaptoethanol, and 1 mM unlabelled IAA. The homogenate was sonicated twice at 10 W for 30 s to disintegrate the protein bodies.
Determination of the high molecular weight IAA-conjugates
5 or 10 µl aliquots of the sonicated homogenate was spotted onto Whatman 3MM or Miracloth filters (15 mm in diameter) followed by immersion in ice-cold 10% trichloroacetic acid containing 0.5 mM unlabelled IAA. Filters were washed with the above solution five times for 15 min each time, followed by 15 min in 95% ethanol containing 1 mM IAA. Radioactivity on the dried filters was determined by scintillation counting. The homogenate, obtained after sonication, was ultracentrifuged at 60 000 g for 30 min. Solid ammonium sulphate was added to the resultant supernatant to obtain a 67% (w/v) saturated solution. The solution was stirred for 1 h and was clarified by centrifugation at 15 000 g for 40 min. The pellet was rinsed twice with 67% (w/v) saturated solution of ammonium sulphate, and the precipitated proteins were then dissolved in 1 ml 25 mM TRIS-HCl buffer, pH 7.6, containing 1 mM IAA and passed through a Sephadex G-15 column (1x20 cm) equilibrated with 25 mM TRIS-HCl, pH 7.6. Cytosolic protein fractions were pooled and concentrated by ultrafiltration using an Amicon Diaflo YM-10 filter. The pellet after ultracentrifugation was rinsed many times and finally resuspended in 25 mM TRIS-HCl buffer, pH 7.6. 10 µl of the concentrated cytosolic proteins and insoluble material was spotted onto Miracloth filters or 50 µl was placed into the vial with 4 ml of scintillation fluid EcoLite (ICN). The radioactivity was measured in a Wallac 1409 liquid scintillation counter.
Alkaline and enzymatic hydrolysis of high molecular weight IAA conjugates
The concentrated cytosolic proteins and insoluble fraction labelled by [2'-14C] IAA were hydrolysed in 1 N NaOH for 1 h at room temperature in order to identify IAA-ester links. After hydrolysis, 10 µl samples were spotted onto Whatman 3MM paper or Miracloth filters followed by immersion in ice-cold 10% trichloroacetic acid containing 0.5 mM IAA. Filters were washed, dried, and radioactivity was determined by scintillation counting. Alternatively, the concentrated cytosolic protein pool as well as insoluble material was hydrolysed enzymatically during the incubation at 30 °C. At the appropriate time, 15 µl samples were separated on TLC plate and indole-containing compounds were detected with the Ehmann reagent or localized based upon the position of chemically synthesized standards. The regions corresponding to free IAA, IAGlc isomers, unidentified compound (RF 0.2), and denatured high molecular compounds were scraped individually from the plate and the radioactivity was measured in a Wallac 1409 liquid scintillation counter.
Qualitative assay of 1-O-IAGlc synthase
A rapid and sensitive qualitative assay of enzyme activity was based upon separation of the substrates and the reaction products by TLC. 1-O-IAGlc synthase activity was determined in a total volume of 8 µl containing 50 mM HEPES-NaOH buffer, pH 7.6, 7.5 mM UDPG, 4 mM IAA, 2.5 mM MgCl2, and 50 mM D-gluconic acid lactone (as an inhibitor of ß-glucosidase). Incubation was for 30 or 60 min at 30 °C, and the reaction products were separated by TLC using the same standard conditions described above. Indole compounds were detected using the Ehmann reagent. The RF of the various compounds was IAA 0.83, 1-O-IAGlc 0.54, and IAInos 0.36.
Quantitative assay of 1-O-IAGlc synthase
The reaction mixture (100 µl) contained 50 mM HEPES-NaOH buffer, pH 7.6, 7.5 mM UDPG, 4 mM IAA, and 0.05 µCi of [2'-14C] IAA (50 mCi mmol–1), 5 mM myo-inositol, 2.5 mM MgCl2, 0.25 mM 2-mercaptoethanol, and 20 µl of IAInos synthase partially purified from maize endosperm (Kowalczyk et al., 2003). IAInos synthase is a trapping system, which converts ready hydrolysable 1-O-IAGlc to the more stable IAInos. The reaction was started by the addition of 20 µl of enzyme solution, and after a 15 or 30 min incubation at 30 °C was stopped by the addition of 0.5 ml 50% (v/v) 2-propanol. Then 0.5 ml of the reaction mixture was transferred to a 2 ml bed volume DEAE-Sephadex (acetate) column in 50% (v/v) 2-propanol. The uncharged products (IAInos and remaining 1-O-IAGlc) not bound to the ion exchanger were eluted with 50% (v/v) 2-propanol to make a total eluate volume of 5 ml. 1 ml was used for radioactivity measurement in a Wallac 1409 liquid scintillation counter.
Determination of ß-glucosidase activity
During 1-O-IAGlc synthase purification, ß-glucosidase was also monitored using p-nitrophenyl-ß-D-glucopyranoside (pNPG) as the chromogenic substrate. The activity was determined by measurement of the quantity of p-nitrophenol released. 50 µl of enzyme solution was added to 50 µl 2.5 mM pNPG in 50 mM HEPES-NaOH, pH 7.6. The reaction mixture was incubated for 20 min at 30 °C, stopped by the addition of 1 ml 0.1 M NaOH, and the released p-nitrophenol measured colorimetrically at 410 nm.
SDS-PAGE and western blotting
SDS-PAGE was performed according to the method of Ogita and Markert (1979) in a Mini Protean II electrophoresis cell (Bio-Rad) using 12% (w/v) resolving gel. The proteins used as molecular mass standards were a 10 kDa Protein Ladder (Gibco). The separated proteins were transferred to a nitrocellulose membrane in buffer containing 50 mM TRIS, 380 mM glycine, 0.1% (w/w) SDS, and 20% (v/v) methanol. The positions of the protein markers were visualized by staining with Ponceau S. The blot was incubated with primary antibodies against IAGlc synthase purified from maize endosperm (Kowalczyk and Bandurski, 1991). The position of IAGlc synthase was detected by an alkaline phosphatase-mediated immunoassaying procedure using goat anti-rabbit-IgG antibodies (Sigma), conjugated to alkaline phosphatase (Harlow and Lane, 1988).
Protein determination
Protein concentration was determined spectrophotometrically at 280 nm or by the Bradford method (Bradford, 1976) using 
-globulin as a standard.
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Detection of 1-O-IA-glucose synthase activity in immature bean seeds
Though ester-linked IAA conjugates are the ubiquitous form of hormone in different tissues of many plants (Sztein et al., 1995, 2000), the enzymes catalysing synthesis of these compounds have been well characterized only in maize endosperm. It was assumed that ester-linked IAA conjugates found in immature bean seeds are synthesized in a manner similar to that of maize endosperm, and that the synthesis of 1-O-IAGlc would be the first step in this pathway. In point of fact, the elementary qualitative assay showed that in the reaction mixture containing UDP-glucose, IAA, myo-inositol, and homogenate of liquid maize endosperm, synthesis of 1-O-IAGlc and IAA-myo-inositol occurs (Fig. 1, lane 2). Only 1-O-IAGlc is synthesized when the partially purified IAGlc synthase from maize endosperm is used in place of the homogenate (Fig. 1, lane 3), but IA-myo-inositol is the sole product when the reaction mixture contains IAGlc synthase with IAInos synthase partially purified from maize endosperm (Fig. 1, lane 4). Using the same reaction mixture and the whole bean seed homogenate, the synthesis of a product having an RF value the same as authentic 1-O-IAGlc (Fig. 1, lane 6) was observed, but not the product that corresponds to IAInos. 1-O-IAGlc, synthesized by been seed homogenate, was identified by comparing its RF value with that of a synthetic standard. A definitive identification as 1-O-IAGlc was made when a partially purified IAInos synthase from maize endosperm was added to the reaction mixture to convert the putative 1-O-IAGlc to IAInos. The synthesis of IA-myo-inositol (Fig. 1, lane 7) demonstrates that immature bean seeds really contain UDP-glucose:IAA glucosyltransferase producing 1-O-IAGlc.
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During the course of the previous experiments it was observed that 1-O-IAGlc is readily hydrolysed by extracts of different tissue at a rate so large that detection of the IAGlc is not possible. In the present experiments it was found that releasing free IAA from the chemically synthesized 1-O-IAGlc is very rapid, supposedly as result of high ß-glucosidase activity present in the whole homogenate of bean seeds (Fig. 2, lane 2). D-gluconic acid lactone and castanospermine, potential ß-glucosidase inhibitors, significantly inhibit IAGlc breakdown (Fig. 2, lanes 4, 5). By contrast with 1-O-IAGlc, the IAInos conjugate is not hydrolysed by bean homogenate (Fig. 2, lane 7). These results indicate that quantitative determination of IAGlc synthase activity in crude homogenates is possible provided that the synthesized 1-O-IAGlc is immediately converted to IAInos using IAInos synthase as a trapping system or that ß-glucosidase inhibitors are present in the reaction medium.
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As shown in Fig. 2 (lanes 3, 4, 5), in addition to the compounds identified on the basis of the RF value or mobility of standards, an unidentified new compound, detectable by the Ehmann reagent, was observed during incubation. This new compound is also produced during incubation of homogenate with IAInos (Fig. 2, lanes 6, 7) or homogenate alone (Fig. 2, lanes 1, 2). On the basis of this result it is assumed that this compound is the peptide containing IAA or tryptophan (positive reaction with the Ehmann and ninhydrin reagent) accumulated as a result of proteolytic degradation of some protein.
The distribution of 1-O-IAGlc synthase in different dicotyledonous plants
Quantitative measurement of IAGlc synthase activity in crude homogenate of some plants was based on the measurement of uncharged reaction products, mainly IAInos (not hydrolysed by tissue extract) synthesized from 1-O-IAGlc in the presence of IAInos synthase partially purified from maize endosperm. Table 1 shows activities (on a fresh weight basis) of the 1-O-IAGlc synthase in crude homogenate of seeds or in vegetative tissue extracts of the examined species. The enzyme activity was assayed in immature seeds of 12 plant species. Activity of 1-O-IAGlc synthase was found in five of the species examined as shown in Table 1. The highest activity of 1-O-IAGlc synthase was found in very young Pisum sativum, Phaseolus vulgaris (Leguminosae), and Brassica napus (Cruciferae) seeds, and Capsella bursa-pastoris fruits. Immature pea seeds (3 mm in diameter) contain IAGlc synthase activity (145 nmol IAGlc min–1 g–1 FW) comparable with the activity of the enzyme present in liquid maize endosperm (142 nmol IAGlc min–1 g–1 FW). During development of pea seeds, the 1-O-IAGlc synthase activity declines rapidly, reaching 8-fold lower activity in the seeds that were 6 mm in diameter. Vegetative tissues of Pisum sativum and young pods from Phaseolus vulgaris display only a trace of 1-O-IAGlc synthase activity. 1-O-IAGlc synthase activity has not been found in immature seeds of watermelon, cucumber, tomato, and maple.
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1-O-IAGlc synthase purification from immature pea seeds
Attempts to purify IAGlc synthase from pea seeds were not entirely successful owing to the lability of partially purified preparation during column chromatography (Fig. 3). Total activity in the homogenate increased 1.8-times after PEG 600 precipitation, however, it drastically dropped during the next chromatography steps. The final enzyme preparation displayed only a trace of 1-O-IAGlc synthase activity (usually no more than 2% of the initial activity in the homogenate) and still contained ß-glucosidase activity, as well as high hydrolytic activity which breaks-down phosphoenolpyruvate (PEP). The presence of this hydrolase in the final preparation interferes with the determination of 1-O-IAGlc synthase activity based on a coupled enzyme assay.
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Immunological cross-reactivity
Anti-maize (1-O-IAGlc synthase) antibody obtained previously by Kowalczyk and Bandurski (1991) was used in the test for immunological cross-reactivity. Partially purified preparations of IAGlc synthase from corn liquid endosperm and immature pea and rape seeds (after DEAE-Sephacel chromatography step) were resolved on 12% polyacrylamide gel. The separated polypeptides, after being blotted onto a nitrocellulose membrane, were tested for cross-reactivity with anti-IAGlc synthase antibody. As shown in Fig. 4, a single immunoreactive band was observed at a locus corresponding to 50 kDa in the case of maize enzyme (Fig. 4, lane 1), as well as single immunoreactive bands of 45.7 and 43.7 kDa with partially purified enzyme preparations from pea and rape, respectively (Fig. 4, lanes 2, 3).
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Transfer of IAA from 1-O-IAGlc to IAA acceptor
The reversible reaction synthesis of 1-O-IAGlc is not favoured energetically owing to the high energy of the acyl bond, but it can be favoured by the relatively high levels of UDP-glucose in the tissue and by the downstream synthesis of IAInos, di-O-(indole-3-acetyl)-D-glucose, and IA-di- or trisaccharides (IAA-sucrose, IAA-oligosaccharides from the raffinose family) as indicated in maize endosperm (Szmidt-Jaworska et al., 1997; Leznicki and Bandurski, 2001). It was expected that the synthesis of 1-O-IAGlc in pea seeds would also be coupled to a downstream reaction, but, as shown here, it is not coupled to the synthesis of IAInos. A possible transfer reaction has been tested using medium containing 1 mM chemically synthesized 1-O-IAGlc and 5 mM disaccharides (melibiose, sucrose, and maltose) or 1 mM [14C] glucose. The reaction was conducted using an homogenate of immature pea seeds that had been pretrated with IAA, as described in the Materials and methods, and the reaction products were separated by TLC. Using qualitative or quantitative ([14C] glucose) determination of the expected products, the synthesis of IAA-conjugates that contain an IAA-moiety originating from 1-O-IAGlc was not observed (data not shown). In these experiments, possible transferase activity was also tested, which would transfer the IAA-moiety from 1-O-IAGlc to aspartic acid, glutamic acid, and leucine. The reaction was conducted for 2 h and 18 h at 30 °C using the immature pea seeds (4 mm in diameter) homogenate, and the reaction products were separated by TLC. Independently of incubation time, the formation of a new IAA-conjugate corresponding to IA-aspartate, IA-glutamate, or IA-leucine was not observed (data not shown).
Labelling of high molecular compounds in pea seeds by [2'-14C] IAA
Bean seeds harvested at full maturity contain some proteins and peptides to which IAA is covalently attached. On the basis of these data it was assumed that 1-O-IAGlc produced in immature seeds can be used for post-translational covalent modification of some proteins. For in vivo protein labelling, 0.5 g of immature pea seeds (3–4 mm) was incubated with labelled IAA as described in the Materials and methods. In a parallel experiment, the incubation medium also contained 0.5 mM unlabelled 1-O-IAGlc. The results shown in Table 2 indicate that material precipitated by TCA in filters contains [2'-14C] IAA, and that the presence of unlabelled 1-O-IAGlc in the incubation medium causes a 19.9–22.4% decrease of the total IAA attached to TCA-precipitated material. These results suggest that at least a part of the IAA bounded to high molecular compounds originated from 1-O-IAGlc synthesized by 1-O-IAGlc synthase.
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Alkaline hydrolysis of the high molecular IAA conjugates
To establish the subcellular localization of these compounds, the homogenate from immature pea seeds incubated with [2'-14C] IAA was ultracentrifuged and the radioactivity was determined in the soluble fraction as well as in the pellet containing the insoluble material. The results of these experiments indicate that IAA is bound to both of these fractions accounting for about 43–49.8% and 50.1–57% in the soluble and insoluble fractions, respectively (Table 3). Ester-linked IAA, contained both in the soluble protein fraction precipitated with ammonium sulphate and in insoluble material, was determined as IAA released from the samples by hydrolysis for 1 h in 1 N NaOH at room temperature. Following hydrolysis, 10 µl samples were spotted onto Whatman 3MM or Miracloth filters and precipitated by TCA. As shown in Table 3, 38–45.6% of the total radioactivity was released from the insoluble fraction, while only 6–9% of the IAA was released from the soluble fraction. Supposedly, this alkali-labile IAA is ester-linked with polysaccharides and glycoproteins or ester-bound with the hydroxyl group present in amino acid residues. The physiological role of high molecular weight IAA conjugates is unknown, although it has been proposed that these compounds can be enzymatically hydrolysed to yield free IAA. On this assumption, the enzymatic hydrolysis of high molecular weight IAA conjugates was assayed in a mixture containing 50 µl of the concentrated soluble protein fraction and 50 µl of the insoluble fraction. After the appropriate time of the incubation, 15 µl samples were separated on a TLC plate and labelled compounds were localized, based upon the position of standards. Each appropriate region was scraped individually and radioactivity was determined in a scintillation counter. As shown in Fig. 5A the formation of the unidentified product giving colour spots with the Ehmann and ninhydrin reagents was observed, however this compound was not [14C] IAA labelled. The accumulation of this supposedly tryptophan-containing peptide or modified tryptophan (lower mobility on TLC than tryptophan) is significantly inhibited by PMSF (results not shown). It is of interest that radioactive compounds increased during the incubation period correspond to free IAA and IAA-glucose isomers (Fig. 5B). Based on these data it is supposed that free IAA released from IAA-labelled high molecular weight compounds is a product of enzymatic hydrolysis of ester- or amide-linked IAA, while the IAA-containing compound, probably IAGlc isomers, is a product of enzymatic hydrolysis of IAA-polysaccharides or IAA-glycoproteins. These results imply that high molecular weight IAA conjugates accumulated in great quantities in the cotyledons of seeds may be considered as the major possible source of IAA required for the growth of seedlings and support the hypothesis that these conjugates are the components of a system for homeostatic control of IAA in plant tissues.
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The widespread distribution and high concentrations of IAA conjugates in plant tissues suggest that these compounds may play an important role in the metabolism of IAA. The physiological significance of the individual conjugate moieties is yet to be determined, but now is generally accepted that different conjugates play different roles in IAA metabolism. The results obtained up to now have suggested the general conclusion that ester conjugates predominate in seeds of monocotyledonous plants (Cohen and Bandurski, 1982; Slovin et al., 1999). Most of the knowledge on ester-linked IAA conjugates comes from studies on maize. The synthesis of IA-glucose, followed by transacylation to myo-inositol represents two potential regulatory steps for the control of IAA concentration by converting hormonally active free IAA into growth-inactive IAA ester. Since all corn tissues hydrolyse IA-glucose isomers and, more slowly, IAInos to free IAA, the mechanism is interpreted as a shuttle to adjust the free pool of IAA via the temporal storage of IAA esters.
Compared with maize, little is known about IAA-conjugate synthesis in other plants. Until now there has been no knowledge about the enzymes that synthesize ester-linked IAA in dicotyledonous plants as well as the enzymes catalysing the synthesis of IAA-amino acids and IAA–protein conjugates. In this report, it is demonstrated that immature seeds of some dicotyledonous plants possess high activity of 1-O-IAGlc synthase, comparable to the activity that was previously investigated in liquid maize endosperm. Western blot analysis demonstrated that partially purified pea and rape enzyme cross-reacts with anti-maize (IA-glucose synthase) antibody. It is notable that the molecular mass of both polypeptides is about 4 and 6 kDa smaller compared with the estimated molecular mass of the maize enzyme (Kowalczyk and Bandurski, 1991; Szerszen et al., 1994). Attempts to purify IAGlc synthase from pea seeds were not entirely successful owing to the lability of the preparation during the purification procedures. 1-O-IAGlc synthase from immature pea, as well as from bean seeds, is extremely unstable and complete loss of activity was found following chromatographic separations. Partially purified pea enzyme indicates the highest activity toward IAA, but significant activity (qualitatively determined) has also been observed toward indole butyric acid and naphthaleneacetic acid. Though IAA is the preferred substrate for pea enzyme in vitro, it is likely that in vivo 1-O-IAGlc synthase may glucosylate either IAA or IBA depending on the relative availability of substrates and the relative compartmentalization of the enzyme and substrates.
It is of interest to elucidate what kind of reaction is coupled with the synthesis of 1-O-IA-glucose which pushes this energetically unfavourable synthesis. These results indicate that, in legume plants (Pisum sativum, Phaseolus vulgaris), the coupled reaction is neither the synthesis of IAInos nor the transfer of the IAA moiety to glucose or some disaccharides. Also, the in vitro experiments indicate that transfer of the IAA-moiety from 1-O-IAGlc to some amino acids does not occur in the pea seed homogenate. However, Delbarre et al. (1994) indicated that tobacco leaf protoplasts incubated with labelled NAA or IAA first accumulate an auxin–glucose ester and only then the auxin–aspartate conjugate. Because it is not known to which downstream reaction the synthesis of 1-O-IAGlc is coupled in pea seeds, attention has been given to the synthesis of some high molecular weight IAA-conjugates. The first such compound was found in bean seeds when Bialek and Cohen (1986) isolated the hydrophobic 3.6 kDa peptide. Recently, a 35 kDa protein encoded by the IAP1 gene has been isolated and cloned (Walz et al., 2002). It was also observed that the presence of the most abundant IAA-modified proteins in bean seeds correlates with a developmental period of rapid growth during seed development and then they are rapidly degraded during germination. Thus, the authors’ idea was that 1-O-IAGlc synthesized in immature legume seeds serves as a donor of the IAA moiety which is then transferred to some proteins, glycoproteins or some high molecular weight polysaccharides. Indeed, the results of in vivo experiments described in the present paper demonstrate that some proteins and perhaps other high molecular weight compounds of pea seeds are labelled in vivo by [14C] IAA. Unlabelled 1-O-IAGlc present in the incubation medium clearly inhibits labelling of high molecular weight compounds The pools of labelled compound were almost equal, both in the soluble and insoluble fractions and accounted for 43–49.8% and 50.1–57%, respectively. While 38–45.6% ester-linked IAA was present in the insoluble fraction, the soluble protein fraction contained only 6–9% ester conjugates as estimated on the basis of the hydrolysis in 1 N NaOH. It can be supposed that the amide-linked IAA represents a significant amount of IAA present in this fraction. Moreover, IAA–protein conjugates enzymatically hydrolysed in vitro yield free IAA and probably IAA–glucose conjugate. It is very likely that the formation of the high molecular weight IAA-conjugates is 1-O-IAGlc dependent and that it is the downstream reaction to which the synthesis of 1-O-IAGlc is coupled in immature seeds of legume plants.
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Acknowledgement |
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The authors wish to thank Professor Robert S Bandurski for his critical reading of this manuscript.
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