Storage lipids as a source of carbon skeletons for asparagine synthesis in germinating seeds of yellow lupine (Lupinus luteus L.)

ARTICLE IN PRESS Journal of Plant Physiology 167 (2010) 717–724

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Storage lipids as a source of carbon skeletons for asparagine synthesis in germinating seeds of yellow lupine (Lupinus luteus L.) S"awomir Borek n, Lech Ratajczak ´ , Poland Department of Plant Physiology, Adam Mickiewicz University, ul. Umultowska 89, 61-614 Poznan

a r t i c l e in fo

abstract

Article history: Received 7 August 2009 Received in revised form 14 December 2009 Accepted 14 December 2009

The 14C-acetate metabolism and regulatory functions of sucrose and sodium fluoride (NaF) were examined in embryo axes and cotyledons isolated from yellow lupine seeds and grown in vitro. After 15 min of incubating organs in solutions of labeled acetate, more radioactivity was found in amino acids (particularly in glutamate, asparagine and glutamine) than in sugars. After 120 min of incubation, 14C was still localized mainly in amino acids (particularly in asparagine and glutamate). The 14C atoms from position C-1 of acetate were mostly localized in the liberated 14CO2, whereas those from position C-2 were incorporated chiefly into amino acids, sugars and the insoluble fraction of the studied organs. The addition of NaF caused a decrease in the amount of 14C incorporated into amino acids and in the insoluble fraction. The influence of NaF on incorporation of 14C into sugars differed between organs. In embryo axes, NaF inhibited this process, but in cotyledons it stimulated 14C incorporation into glucose. The release of 14CO2 with the C-1 and C-2 carbon atoms from acetate was more intensive in embryo axes and cotyledons grown on a medium without sucrose. This process was markedly limited by NaF, which inhibits glycolysis and gluconeogenesis. Alternative pathways of carbon flow from fatty acids to asparagine are discussed. & 2010 Elsevier GmbH. All rights reserved.

Keywords: Acetate Carbon flow Mobilization of storage lipids Sodium fluoride Sucrose

Introduction Storage lipid mobilization has been studied exclusively in germinating seeds of oil plants. There is less published information about this process in seeds that are rich in protein or starch, but contain much lower amounts of storage lipids. In germinating oil-storing seeds, lipid mobilization can be divided into several steps, including b-oxidation, glyoxylate cycle, tricarboxylic acid cycle, and gluconeogenesis (Beevers, 1976, 1979; Mettler and Beevers, 1980; Weir et al.; 1980; Allen et al., 1988; Comai et al., 1989; Turlay and Trelease, 1990; De Bellis and Nishimura, 1991; Rylott et al., 2001; Penfield et al., 2005; Penfield et al., 2006; Graham, 2008; Quettier and Eastmond, 2009). Aconitase, one of the enzymes of the glyoxylate cycle, is active in the cytosol but not in glyoxysome (Courtois-Verniquet and Douce, 1993; De Bellis et al., 1994, 1995; Hayashi et al., 1995; Cots and Widmer, 1999; Eastmond and Graham, 2001; Kunze et al., 2006; Graham, 2008; Pracharoenwattana and Smith, 2008). Aconitase transforms citrate into isocitrate, which can then be transported to the glyoxysome where it becomes the substrate for isocitrate lyase. In this way, the flow of carbon from fatty acids to carbohydrates may

Abbreviations: IDH, isocitrate dehydrogenase; GOGAT, glutamate synthase; GS, glutamine synthase; NaF, sodium fluoride; MDH, malate dehydrogenase n Corresponding author. Tel.: + 48 61 8295893; fax: + 48 61 8295887. E-mail address: [email protected] (S. Borek). 0176-1617/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2009.12.010

proceed according to the pathway proposed by Beevers (1976, 1979) and Mettler and Beevers (1980). However, high activity of NADP + -dependent isocitrate dehydrogenase (IDH) is observed in the cytosol (Nieri et al., 1995; Chen, 1998; Falk et al., 1998; ¨ Hodges et al., 2003; Igamberdiev and Gardestrom, 2003; Kihara et al., 2003). NADP + -dependent IDH transforms isocitrate into 2oxoglutarate and is active in germinating seeds of yellow lupine (Borek et al., 2006). The role of lipids as suppliers of isocitrate for cytosolic IDH has been reported previously by Nieri et al. (1995). Isocitrate produced in the cytosol may participate in other metabolic pathways, outside glyoxysome. After oxidation, it provides carbon skeletons for amino acid synthesis, because the product of cytosolic IDH, i.e. 2-oxoglutarate, can be transaminated with various amino acids, resulting in formation of glutamate (Hodges et al., 2003). This amino acid plays a key role in the metabolism of plant cells, especially in the glutamine synthase/ glutamate synthase (GS/GOGAT) cycle, which is the main cycle of primary amino acid biosynthesis. This hypothesis sheds light on the possibility of cooperation between carbon and nitrogen compounds in plant metabolism. It is necessary to verify the idea that the tricarboxylic acid cycle (aided by the anaplerotic malate pathway) is the major source of carbon skeletons for amino acid metabolism. The results of experiments with germinating yellow lupine seeds indicate that the amino acids metabolism is linked with the pathway of degradation of fatty acids released during triacylglycerol hydrolysis. It appears that in legumes, storage

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lipids may serve not only as substrates for respiration and gluconeogenesis, but may also be utilized to fuel the synthesis of nitrogen compounds (Borek et al., 2003). Another important and still unexplained issue is the control of storage lipid mobilization during seed germination and the relationships between carbon metabolism and nitrogen metabolism. Some reports indicate that the factor controlling the intensity of storage lipid mobilization is sugar, i.e. the end product of storage lipid mobilization. Exogenous sucrose (20 mM) causes a slight delay in germination of Arabidopsis thaliana seeds (Rylott et al., 2001), but a higher sucrose concentration (330 mM) inhibits germination and seedling growth (Laby et al., 2000). A concentration of 110 mM of exogenously applied glucose significantly retards mobilization of seed storage lipids in Arabidopsis seedlings, and this effect is not due to osmotic stress (To et al., 2002). The expression of genes encoding the two ‘‘marker enzymes’’ of the glyoxylate cycle (isocitrate lyase and malate synthase) was found to be induced under a critical deficiency of sucrose, mannose, and fructose. Exogenous addition of any of those sugars (as well as 2-deoxyglucose) to the growth medium caused inhibition of expression of the genes encoding both enzymes (Graham et al., 1994). Activity and gene expression of acyl-CoA oxidase (b-oxidation), isocitrate lyase, and malate synthase (glyoxylate cycle), and phosphoenolpyruvate carboxykinase (gluconeogenesis) in germinating seeds and seedlings of Arabidopsis is tightly coordinated both within and between the major pathways of b-oxidation, the glyoxylate cycle, and gluconeogenesis. This regulation is mainly transcriptional and might occur by a global regulatory mechanism (Rylott et al., 2001). It is not known whether the regulation of storage lipid mobilization in non-oil plants is the same as in A. thaliana. This is particularly important in legumes, whose seeds contain large amounts of storage proteins. For example, seeds of Lupinus luteus L. and Lupinus mutabilis Sweet contain up to 35% of their dry weight as storage proteins (Lehmann and Ratajczak, 2008). Lipid content in seeds of L. luteus L. reaches about 6% and in seeds of L. mutabilis Sweet, about 20% of the dry weight (Borek et al., 2009). In germinating lupine seeds, amino acids from storage protein mobilization are used not only for synthesis of new proteins, but also are partially used as respiratory substrates. Nitrogen liberated during amino acids oxidation is stored in asparagines, which can be accumulated in large quantities in germinating lupine seeds and may make up to 30% of the dry matter (Lehmann and Ratajczak, 2008). The source of carbon skeletons for such intensive asparagine synthesis is not known. Matured, air-dried yellow lupine seeds do not contain starch as a storage compound (M"odzianowski and Weso"owska, 1975; Hoffmannowa and Zielin´ska, 1981; Borek et al., 2006), so carbon skeletons for asparagine synthesis do not come from carbohydrates. Storage lipids may be a source of carbon skeletons for asparagine synthesis in germinating lupine seeds. In experiments on storage lipid catabolism in germinating seeds, exogenously applied acetate is used as the simplest fatty acid. It is possible that exogenous acetate may be directly transported into mitochondrion and oxidized in the TCA cycle, but in Arabidopsis seedlings, a portion of exogenously applied acetate is directed into glyoxysome, where in glyoxylate cycle, it is converted to other acids (malate, citrate or succinate) and is translocated in this form to mitochondria (Eastmond et al., 2000; Hooks et al., 2004; Turner et al., 2005; Kunze et al., 2006; Hooks et al., 2007). Experiments carried out on acetate non-utilizing Arabidopsis acn1 and acn2 mutants have shown that exogenous acetate is transported into peroxisome through translocator COMATOSE (CTS), which is a member of the ABC (ATP-binding-cassette) superfamily of proteins (Kunze et al.,

2006; Hooks et al., 2007). In peroxisomes, acetate is activated by acetyl-CoA synthetase (AcetCS) (Turner et al., 2005; Hooks et al., 2007), which is more active with acetate than with butyrate and is not active with fatty acids longer than C-4 (Turner et al., 2005). Acetyl-CoA is partitioned between malate synthase and citrate synthase in the glyoxylate cycle (Hooks et al., 2007). To explore the pathways of fatty acid catabolism in germinating lupine seeds, isolated embryo axes and excised cotyledons were incubated in medium containing acetate specifically labeled with 14C at positions C-1 or C-2. Incorporation of 14C into sugars, amino acids, and released 14CO2 was analyzed (according to Beevers, 1991). The regulatory function of sucrose on these processes was studied, and the effect of sodium fluoride (NaF) was also investigated. NaF is a strong inhibitor of enolase (phosphoenolpyruvate hydratase) (Miller, 1993; Qin et al., 2006), so it is considered as a factor limiting respiration already at the glycolysis stage. NaF inhibits gluconeogenesis at the enolase step as well. Moreover, NaF is an inhibitor of H + -ATPase (Miller, 1993; Fac- anha and de Meis, 1995; Reddy and Kaur, 2008). While inhibition of enolase is based on direct enzyme inhibition (Qin et al., 2006), the inhibition of H + -ATPase is considered as a result of the binding of fluoride to Ca2 + and Mg2 + , which decreases availability of these ions to enzymes (Reddy and Kaur, 2008). By applying sucrose and NaF, 14C-acetate metabolism and regulatory functions of sucrose and sodium fluoride (NaF) were examined in embryo axes and cotyledons isolated from yellow lupine seeds and grown in vitro.

Material and methods Plant material and growth conditions Seeds of L. luteus L. were surface sterilized in 0.02% mercuric chloride for 10 min and allowed to imbibe for 24 h at 25 1C. Isolated from imbibed embryo axes and cotyledons were grown in vitro for 96 h on Heller (1954) medium supplemented with 60 mM sucrose ( + S) or without the sugar ( S). The in vitro culture was kept in the dark at 25 1C. Radiolabel experiments Embryo axes and cotyledons from in vitro cultures were incubated in Heller medium ( + S and S) containing 925 kBq of 1or 2-14C-acetate, as described previously (Borek et al., 2003). The incubation mixture was also supplemented with NaF in one variant of the experiment. The concentration of NaF was adjusted so that after 120 min of incubation of the studied organs with radiolabeled acetate, their respiration rate decreased by 50%. This was performed by a Clark electrode (Rank Bothers, Digital Model 10). The final concentration of NaF reached 30 mM for embryo axes and 35 mM for cotyledons. The radioactivity of liberated CO2 was measured over 120 min of incubation, and after 15 and 120 min of incubation, radioactive carbon atoms in selected sugars and amino acids were localized with the use of descending paper chromatography and as described previously (Borek et al., 2003).

Results Catabolism of 1- and 2-14C-acetate in embryo axes and cotyledons A measure of the catabolism of 1- and 2-14C-acetate was the radioactivity of released CO2 over 120 min of incubation. The highest amount of 14CO2 containing the carbon atom from both positions of acetate was observed in the 30th minute of the

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Fig. 1. (A–D) Radioactivity of CO2 with 14C from 1-14C- and 2-14C-acetate released by embryo axes and cotyledons grown for 96 h in vitro on medium with 60 mM sucrose (+ S) or without sucrose ( S) and incubated for 120 min with 1-14C- or 2-14C-acetate and sodium fluoride (+ NaF) or without the fluoride ( NaF). The data are means7 SD of three independent experiments.

120 min incubation (Fig. 1A–D). The embryo axes grown on the medium without sucrose ( S) released 14CO2 with the carbon atom from position C-1 more intensively than those grown on the medium with sucrose (+ S) (Fig. 1A). The carboxyl 14C atom of acetate was also incorporated into CO2 by cotyledons, but at a much lower level (Fig. 1B). The dynamics of the release of 14CO2 with the carbon atom from position C-2 by embryo axes and cotyledons was very similar to that of the release of 14CO2 with the carbon atom from position C-1, but at a much lower level (Fig. 1A and B). Sodium fluoride (NaF) caused a decrease in the release of 14CO2 with carbon atoms from both positions of acetate by embryo axes and cotyledons. The difference in 14CO2 release by embryo axes between + S and S variants of in vitro culture was less conspicuous than during incubation of embryo axes and cotyledons without NaF (Fig. 1A–D).

Radioactivity distribution among sugars, amino acids and the insoluble fraction of axes and cotyledons The incorporation of 14C from acetate into selected sugars, amino acids and the insoluble fraction of the isolated organs was generally greater from the C-2 position than from C-1 position. This was observed after 15 (Table 1) and 120 min of incubation (Tables 2–4) in media containing radiolabeled acetate. After 15 min of incubation, radioactive sugars (sucrose, glucose, and fructose) were not found in the embryo axes grown on the medium without sucrose ( S). In cotyledons, the radioactivity of the sugars (from both C-1 and C-2 positions) was similar. Radioactivity levels of amino acids in almost all cases were markedly higher than those of sugars (Table 1). The highest

radioactivity was recorded for glutamate in embryo axes; however, in cotyledons, glutamate radioactivity was very low. The second highest radioactivity was noted for asparagine. Surprisingly, no radioactivity was recorded for its precursor, i.e. aspartate (both C-1 and C-2 positions) in embryo axes (Table 1). After 120 min, the incorporation of 14C from acetate in embryo axes fed with sucrose (+ S) was most intensive into glucose. In starved ( S) embryo axes, no radiolabeled sugars were detected (Table 2). The radioactivity of amino acids after 120 min of incubation was much higher (with few exceptions) in organs grown on the + S medium than on the S medium. The highest level of radioactivity was recorded in the same amino acids as after 15 min of incubation, i.e. in amino acids of the primary amino acid biosynthesis pathway (Table 3). Moreover, arginine and lysine radioactivity was high in embryo axes. Cotyledons from both trophic variants of in vitro culture ( + S and S) were characterized by effective incorporation of 14C from both positions into aspartate, whereas in embryo axes, no radioactivity was detected in aspartate (Table 3). NaF markedly decreased the incorporation of 14C from both positions of acetate into sucrose (Table 2), arginine, lysine, aspartate, asparagine, glutamate, glutamine (Table 3), and the insoluble fraction (Table 4). The inhibiting effect of NaF on incorporation of 14C from position C-2 into amino acids in embryo axes and cotyledons was stronger on the +S medium. The inhibition of incorporation of 14C from position C-1 was similar, but the result was less evident (Table 3). The radioactivity of the insoluble fraction of embryo axes and cotyledons was markedly higher in organs cultured on the +S medium than on the S medium. However, NaF limited the incorporation of 14C from both positions of acetate in a much stronger way on the +S medium than on the S medium (Table 4).

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Table 1 Incorporation of 14C from 1-14C- and 2-14C-acetate into selected sugars, amino acids, and insoluble fraction of isolated embryo axes and excised cotyledons grown for 96 h in vitro on a medium without sucrose ( S). Organs were incubated with labeled acetate for 15 min. The data are means of three independent experiments. SD does not exceeds 10%. 1-14C incorporation (CPM g

Total radioactivity of the sample (103) Sucrose Glucose Fructose Total radioactivity of the sample (103) Arginine + lysine Aspartate Asparagine Glutamate Glutamine Alanine Methionine + valine Phenylalanine Leucine+ isoleucine Radioactivity of insoluble fraction (103)

1

2-14C incorporation (CPM g

FW)

1

FW)

Embryo axes

Cotyledons

Embryo axes

Cotyledons

8 0 0 0 5 323 0 665 1349 428 350 215 256 399 12

15 176 136 183 10 47 106 779 115 118 269 214 295 94 4

11 0 0 0 7 386 0 855 2050 628 678 345 372 482 14

16 207 145 210 11 40 182 519 134 196 319 253 341 138 4

Table 2 Incorporation of 14C from 1-14C- and 2-14C-acetate into selected sugars in embryo axes and cotyledons grown for 96 h in vitro on medium with 60 mM sucrose (+ S) or without sucrose ( S) and incubated for 120 min with 1-14C- or 2-14C-acetate and sodium fluoride (+ NaF) or without the fluoride ( NaF). The data are means of three independent experiments. SD does not exceeds 10%. 1-14C incorporation (CPM g

1

Embryo axes +S NaF 3

Total radioactivity of the sample (10 ) Sucrose Glucose Fructose

80 1465 2600 365

Cotyledons S

+ NaF 55 666 440 256

2-14C incorporation (CPM g

FW)

NaF 26 0 0 0

Embryo axes

+S + NaF 13 0 0 0

NaF 197 650 399 650

S +NaF 38 440 900 490

1

NaF 101 503 300 550

+S + NaF 43 359 1003 647

NaF 94 1899 2707 760

Cotyledons S

+ NaF 44 625 510 169

FW)

NaF 34 0 0 0

+S + NaF 29 0 0 0

NaF 206 899 430 730

S + NaF 54 870 1100 820

NaF 169 880 700 799

+NaF 65 630 1089 852

Table 3 Incorporation of 14C from 1-14C- and 2-14C-acetate into selected amino acids in embryo axes and cotyledons grown for 96 h in vitro on a medium with 60 mM sucrose ( +S) or without sucrose ( S) and incubated for 120 min with 1-14C- or 2-14C-acetate and sodium fluoride (+ NaF) or without the fluoride ( NaF). The data are means of three independent experiments. SD does not exceeds 10%. 1-14C incorporation (CPM g-1 FW)

2-14C incorporation (CPM g-1 FW)

Embryo axes

Embryo axes

+S NaF 3

Total radioactivity of the sample (10 ) 54 Arginine + lysine 1500 Aspartate 0 Asparagine 2752 Glutamate 6514 Glutamine 2200 Alanine 427 Methionine + valine 0 Phenylalanine 458 Leucine+ isoleucine 0

Cotyledons S

+ NaF 37 285 345 200 666 730 350 402 190 330

NaF 17 1048 0 3002 5559 1800 260 0 492 0

+S + NaF 7 248 248 465 500 721 300 332 200 320

NaF 131 666 2300 13,804 8652 829 300 525 222 499

Discussion The results of experiments with the radiolabeled acetate showed that, in germinating yellow lupine seeds, exogenous acetate is oxidized in the TCA cycle (Fig. 1A–D), but some quantities of 14C coming from acetate are incorporated in amino acids and sugars (Tables 1–3). Radioactive amino acids in the

S +NaF 25 165 460 301 399 190 240 301 267 159

NaF 67 439 1121 4111 4555 700 223 267 179 260

+S +NaF 28 230 1199 250 300 301 299 282 152 222

NaF

Cotyledons S

+ NaF

63 29 3526 193 0 235 5902 172 17,086 1000 3666 942 1200 331 0 685 650 260 0 765

NaF 23 2440 0 6101 11,000 1932 611 0 550 0

+S +NaF 19 500 2120 1150 1980 2221 1801 1840 2030 718

NaF 137 680 2411 14,498 13,959 930 550 865 295 862

S +NaF 36 162 559 201 367 299 867 244 320 252

NaF 113 600 1499 8951 8200 801 673 628 200 701

+NaF 43 270 811 528 509 377 601 200 210 340

embryo axes and cotyledons were identified already after 15 min of incubation. After 15 and 120 min of incubation, the majority of 14 C atoms from radiolabeled acetate were incorporated into amino acids belonging to the primary amino acid biosynthesis pathway, i.e. glutamate, asparagine, glutamine, and aspartate (Tables 1 and 3). Among these amino acids, the highest radioactivity after 15 min of incubation was found for glutamate and

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Table 4 Incorporation of 14C from 1-14C- and 2-14C-acetate into insoluble fraction of embryo axes and cotyledons grown for 96 h in vitro on a medium with 60 mM sucrose (+ S) or without sucrose ( S) and incubated for 120 min with 1-14C- or 2-14C-acetate and sodium fluoride (+ NaF) or without the fluoride ( NaF). The data are means of three independent experiments. SD does not exceeds 1%. 1-14C incorporation (CPM 103 g

1

Embryo axes +S NaF 192

Cotyledons S

+ NaF 11

2-14C incorporation (CPM 103 g

FW)

NaF 87

14

NaF 49

FW)

Embryo axes

+S + NaF

1

S + NaF 7

NaF 23

+S + NaF 13

asparagine in embryo axes, and asparagine in cotyledons (Table 1), and after 120 min in asparagine and glutamate, both in embryo axes and cotyledons (Table 3). Moreover, after 15 and 120 min of incubation, the radioactivity of the amino acids, with a few exceptions, markedly exceeded the radioactivity of sugars (Tables 1–3). The results presented in this paper show the specificity of legumes. Considering the domination of the asparagine synthesis pathway in legume seedlings (Lehmann and Ratajczak, 2008), the carbon and nitrogen metabolism may be linked in a number of different ways. In germinating legume seeds, it is necessary to utilize the large quantities of ammonia produced in the course of storage protein mobilization, so large amounts of asparagine (up to 30% of seeds dry weight) are accumulated in germinating lupine seeds as a temporary storage form of ammonia (Lever and Butler, 1971; Ratajczak et al., 1990; Lehmann and Ratajczak, 2008). One source of carbon skeletons for asparagine synthesis may be fatty acids coming from storage lipids degradation. In Arabidopsis seedlings, some of the exogenously applied acetate is directed into glyoxysome, where it is activated to acetyl-CoA and is introduced into the pathways of storage lipid catabolism (Turner et al., 2005; Kunze et al., 2006; Hooks et al., 2007). It is highly possible that the same or similar mechanism operates in lupine seedlings. In lupine organs grown in vitro for 96 h, lipase is very active and degradation of oil bodies occurs (especially in organs grown on medium deprived of sucrose; S) (Borek et al., 2006). Because intensive lipid degradation occurred in the developmental stage of lupine seedlings used in experiments with radiolabeled acetate, it is highly possible that exogenous acetate was channeled into pathways of fatty acid catabolism, and the results presented in this paper reflect storage lipid catabolism in lupine seedlings. Many alternative pathways of storage lipid catabolism during seed germination are known (Graham, 2008). In a ‘‘classic’’ pathway, based on experiments with oil-storing seeds, fatty acids are converted to sugars through b-oxidation, the glyoxylate cycle, the tricarboxylic acid cycle, and gluconeogenesis (Beevers, 1976, 1979; Mettler and Beevers, 1980; Weir et al., 1980; Allen et al., 1988; Comai et al., 1989; Turlay and Trelease, 1990; De Bellis and Nishimura, 1991; Rylott et al., 2001; Penfield et al., 2005; Penfield et al., 2006; Graham, 2008; Quettier and Eastmond, 2009). This pathway is active in germinating lupine seeds as well. Evidence of this includes the incorporation of 14C of acetate into sugars in cotyledons already after 15 min of incubation (Table 1) and more intensive radioactivity of CO2 with C-1 than C-2 of acetate (Fig. 1). Succinate is transported from glyoxysome to mitochondria, but in the TCA cycle it is metabolized partially and is exported to cytosol as malate. In germinating lupine seeds, there is a branch in the pathway of lipid conversion to sugars. This branch may occur at the oxaloacetate stage and directs carbon skeletons to asparagine synthesis (Fig. 2A). Carbon flow through this branch is very

NaF 257

Cotyledons S

+ NaF 15

NaF 108

+S + NaF 24

NaF 57

S + NaF 12

NaF 49

+NaF 14

effective because the radioactivity of amino acids was higher than the radioactivity of sugars, both after 15 (Table 1) and 120 min of incubation (Tables 2 and 3). In germinating oil-storing seeds, the modifications of this process are well known, for example in mutants without a glyoxylate cycle (Eastmond et al., 2000; Cornah et al., 2004). Based on this observation, other modifications of the ‘‘classical’’ pathway are possible. Lack of aconitase in glyoxysome (Courtois-Verniquet and Douce, 1993; De Bellis et al., 1994, 1995; Hayashi et al., 1995; Cots and Widmer, 1999; Eastmond and Graham, 2001; Kunze et al., 2006; Graham, 2008; Pracharoenwattana and Smith, 2008) forces translocation of citrate to cytosol and subsequent import of isocitrate to glyoxysome. However, citrate may be directed into mitochondria (Pracharoenwattana et al., 2005), where it is converted into malate. Malate is translocated to cytosol and converted into oxaloacetate, which is transported to the glyoxysome, where it is used to sustain the glyoxylate cycle (Eastmond and Graham, 2001; Baker et al., 2006; Pracharoenwattana et al., 2007; Pracharoenwattana and Smith, 2008). This is a shuttle mechanism that involves glyoxysomal malate dehydrogenase (MDH), which facilitates NADH reoxidation, because NADH is generated in b-oxidation and in the glyoxylate cycle as well (Graham and Eastmond, 2002; Baker et al., 2006; Kunze et al., 2006; Pracharoenwattana et al., 2007; Pracharoenwattana and Smith, 2008). However, in germinating yellow lupine seeds, oxaloacetate may be transaminated in cytosol to aspartate (Ratajczak et al., 1998a), i.e. a precursor of asparagine (Fig. 2B). The highest level of NADH re-oxidation may be achieved by another shuttle mechanism, differing from citrate-oxaloacetate. Glyoxysomal MDH can work in an opposite way from the glyoxylate cycle, i.e. it can convert oxaloacetate to malate and simultaneously oxidize NADH (Baker et al., 2006; Pracharoenwattana et al., 2007; Graham, 2008; Pracharoenwattana and Smith, 2008). Malate is exported from glyoxysome to cytosol and/ or the mitochondrion and is converted again to oxaloacetate by cytosolic and/or mitochondrial MDH. Oxaloacetate is then translocated into the glyoxysome. This is a third possibility for a shuttle mechanism leading to NADH re-oxidation (Mettler and Beevers, 1980; Graham, 2008; Pracharoenwattana and Smith, 2008). However, oxaloacetate in cytosol may be aminated by aspartate aminotransferase (Ratajczak et al., 1998a). Aspartate is a direct precursor of asparagine, and since it is synthesized in cytosol, it may be a substrate for asparagine synthase (Fig. 2C). Evidence for this pathway comes from the lack of radioactive aspartate after 15 min (Table 1) and low radioactivity of this amino acid after 120 min of incubation (Table 3). The fourth alternative pathway of amino acid synthesis from lipids is the reaction catalyzed by active cytosolic NADP + dependent isocitrate dehydrogenase (IDH) in germinating lupine seeds. The activity of cytosolic IDH in germinated yellow lupine

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Fig. 2. (A–D) Schematic representation of the alternative pathways involved in storage lipid mobilization leading to amino acids in germinating seeds of yellow lupine. MDH, malate dehydrogenase.

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seeds is considerably higher in cytosol than in mitochondria (Borek et al., 2006). Citrate is exported to cytosol, converted to isocitrate by aconitase, and may be a substrate for NADP + dependent IDH and be drawn from the ‘‘classic’’ pathway originally proposed by Mettler and Beevers (1980). Isocitrate may be oxidized to 2-oxoglutarate, i.e. a compound that undergoes transamination and/or takes part in the GS/GOGAT cycle, yielding glutamate (Sood et al., 1998; Palomo et al., 1998). Glutamate plays an important role in plant cell metabolism, as it is a precursor for many compounds. Glutamate also plays a key role in the biosynthesis of primary amino acids (Bewley and Black, 1994), including asparagine (Fig. 2D). Glutamate may be synthesized from 2-oxoglutarate coming from the TCA cycle as well. Intermediates of the TCA cycle may be supplemented by citrate or malate. Malate coming from the glyoxysome may be introduced to the TCA cycle by mitochondrial MDH. This anaplerotic supplementation of the TCA cycle allows the channeling of 2oxoglutarate to glutamate synthesis in cytosol. Storage protein degradation in germinating lupine seeds caused an increase in transamination processes, where 2-oxoglutarate plays a central role because it can be an acceptor of amine groups from all of the amino acids (Lehmann and Ratajczak, 2008). The proposed direction of biochemical transformation is also confirmed by the high radioactivity of the released 14CO2 with carbon atoms from position C-1 of acetate. The most intensive release of 14CO2 was observed up to the 30th minute of the 120 min incubation in the solution of radiolabeled acetate (Fig. 1A and B). This likely resulted from oxidation of acetate in the tricarboxylic acid cycle. Nevertheless, the markedly higher release of 14CO2 with carbon atoms from position C-1 of acetate (compared to 14CO2 with carbon atoms from position C-2) indicates that it is formed not only during the tricarboxylic acid cycle within mitochondria, but also during other biochemical processes, such as those catalyzed by cytosolic NADH + –IDH. Another source of 14CO2 may be phosphoenolpyruvate carboxykinase, for which activity in germinating yellow lupine seeds has been detected as well (Ratajczak et al., 1998b). However, lower radioactivity of sugars than amino acids (Tables 1–3) suggests restricted carbon flow through phosphoenolpyruvate carboxykinase. The release of 14CO2 with carbon atoms from position C-2 may be attributed only to reactions of the tricarboxylic acid cycle. Evidence for significance of IDH activity in carbon flow from fatty acids to amino acids includes high radioactivity of glutamate in embryo axes and cotyledons (Tables 1 and 3). It is worth noting that, in embryo axes after 15 and 120 min of incubation, high radioactivity of asparagine was found, but no radioactivity was recorded for aspartate (Tables 1 and 3). This is surprising because aspartate is a substrate for asparagine synthesis. How do we explain the lack of radioactivity of the asparagine precursor? It is likely the result of asparagine synthase operation. In early developmental stages of lupine seedlings, very high activity of asparagine synthase was noted, so aspartate synthesized in cytosol is immediately aminated to asparagine (Lehmann and Ratajczak, 2008). The incorporation of 14C into sugars after 120 min of incubation was more intensive in embryo axes fed with sucrose ( + S), whereas in cotyledons, more radiolabeled sugars (especially with 14 C atoms from position C-2 of acetate) were found in organs grown on the S medium (Table 2). This indicates that sucrose controls the transfer of carbon skeletons to sugar synthesis. In cotyledons, the controlling function of sucrose is not as obvious as in isolated embryo axes. In excised cotyledons, there is no transfer of products of storage compound mobilization to the embryo axis. Accumulation of these compounds inhibits further mobilization of reserves and decreases metabolic activity, so the addition of exogenous sucrose has a limited effect on cell metabolism. Such

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an interpretation is confirmed by the lack of substantial difference in 14CO2 release by excised cotyledons growing on the +S and S media. Sucrose added to the medium markedly intensified the incorporation of 14C into amino acids in the embryo axis and cotyledons in the 120th minute of incubation (Table 3). This result suggests that, in tissues fed with sucrose, amino acids are not substrates for respiration, as the exogenous sucrose is used for this purpose. By contrast, in organs grown on the S medium, the lower level of radioactivity in amino acids could be due to higher utilization of amino acids as respiratory substrates, or to the use of acetate in respiration. Such a sequence of events is also confirmed by greater release of 14CO2 by organs grown on the S medium (Fig. 1A and B), the lower level of radioactivity of the insoluble fraction of organs (Table 4), and the lack of radioactive sugars in embryo axes grown on the S medium (Table 2). This is consistent with the hypothesis that a catabolic repression mechanism exists, which facilitates the saving of organic compounds other than sugars (e.g. for amino acid synthesis) due to the utilization of exogenous sucrose as an energy source (Yu, 1999; Rolland et al., 2006). Higher radioactivity of sugars (Table 2), amino acids (Table 3), and insoluble fraction (Table 4) in organs fed with sucrose is not a result of stimulated acetate uptake. Acetate uptake by organs grown on –S media was probably equal to uptake by organs fed with sucrose because radioactivity of CO2 was higher in –S variants (Fig. 1A and B). Thus, differences in radioactivity among sugars, amino acids, the insoluble fraction and CO2 were not the result of different uptake of acetate, but result from different metabolism of acetate in organs grown on –S and + S media. In lupine embryo axes, the pathways shown in Fig. 2A and B are not fully functional because of low amounts of storage compounds (Borek et al., 2006). This is clearly visible in embryo axes grown on S medium, in which no radioactive sugars were detected (Table 2). Sugar deficiency in tissues of intensively growing organs excludes the conversion of fatty acids to sugars. Under these conditions, the role of mitochondria is magnified. Carbon skeletons from spare amounts of fatty acids (as well as other storage compounds) are oxidized in the tricarboxylic acid cycle as respiratory substrates, and no radioactivity from acetate is incorporated into sugars. The pathways proposed in Fig. 2A and B are fully functional only in cotyledons. NaF markedly reduced the incorporation of 14C from acetate (both C-1 and C-2) into sugars (Table 2), amino acids (Table 3), insoluble fraction (Table 4), and the release of 14CO2 (Fig. 1A–D) by embryo axes and cotyledons. Moreover, NaF inhibited the incorporation of 14C into amino acids more strongly than into sugars, especially on the + S medium. The marked decrease in incorporation of 14C atoms into amino acids, caused by the reduction of the respiratory efficiency of organs under the influence of NaF (Miller, 1993), suggests that the transfer of carbon skeletons from fatty acids to amino acids, leading through mitochondria, is more intensive than the cytosolic pathways proposed in Fig. 2C and D. At this step of experimentation, however, it is impossible to precisely determine insensitivity of carbon flow through the four pathways proposed above (Fig. 2A– D). Most likely, each is functional during lupine seeds germination and they are not mutually exclusive. However, it was shown here that carbon skeletons coming from storage lipid catabolism may, at least partially, supplement the pathway of asparagine synthesis, which is very intensive in germinating lupine seeds.

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