ARTICLE IN PRESS Journal of Plant Physiology 163 (2006) 1061—1070
www.elsevier.de/jplph
Role of asparagine and asparagine synthetase genes in sunflower (Helianthus annuus) germination and natural senescence Marı´a Begon ˜a Herrera-Rodrı´gueza, Jose ´ Marı´a Maldonadob, Rafael Pe ´rez-Vicentec, a
`rea de Fisiologı´a Vegetal, Universidad Pablo de Olavide, Departamento de Ciencias Ambientales, A Ctra. de Utrera, km 1, 41013 Seville, Spain b Departamento de Biologı´a Vegetal y Ecologı´a, Unidad de Fisiologı´a Vegetal, Facultad de Biologı´a, Universidad de Sevilla, Avda. Reina Mercedes 6, 41012 Seville, Spain c `rea de Fisiologı´a Vegetal, Universidad de Co Departamento de Biologı´a Vegetal, A ´rdoba, Edificio C4, Campus de Rabanales, 14071 Co ´rdoba, Spain Received 27 July 2005; accepted 24 October 2005
KEYWORDS Asparagine synthetase; Germination; Natural senescence; Nitrogen mobilization
Summary Sunflower (Helianthus annuus) contains three active asparagine synthetase (EC 6.3.5.4, AS) genes: HAS1, HAS1.1 and HAS2. Asparagine content and AS gene expression were determined during germination and leaf and cotyledon natural senescence to assess the role of asparagine as well as the extent of participation of each AS gene in different nitrogen mobilizing processes. Asparagine accumulated in the dry seed and was the predominant amide throughout germination. During cotyledon senescence, the asparagine level was slightly higher than that of glutamine. The opposite was true for leaf senescence. According to transcript accumulation data, most of the asparagine newly synthesized for germination and cotyledon expansion was due to HAS2 activity, with little contribution of the other AS genes. However, all three genes work together to synthesize asparagine for leaf senescence. The absence of significant AS gene expression in cotyledon senescence differentiates leaf and cotyledon senescence, and suggests a cotyledon-specific regulation. & 2005 Elsevier GmbH. All rights reserved.
Corresponding author. Fax: +34957218939.
E-mail address:
[email protected] (R. Pe´rez-Vicente). 0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2005.10.012
ARTICLE IN PRESS 1062
Introduction Germination and senescence are physiological processes that imply a great mobilization of nitrogen. Reserve protein stored in cotyledons or endosperm is degraded during germination, and their amino acids converted to compounds that are exportable to the growing axis. In leaf senescence, the photosynthetic apparatus is disassembled and its nitrogen incorporated to nitrogen carrier molecules toward its recycling in the plant. Asparagine has been reported to play a prominent role as a nitrogen carrier in nitrogen mobilization during germination of Lupinus albus (Lea and Miflin, 1980), Vigna radiata (Kern and Chrispeels, 1978) and Gossypium hirsutum (Capdevila and Dure, 1977). In pea germination, however, there is no comparable synthesis of asparagine (Larson and Beevers, 1965), showing that the degree of asparagine involvement in germination depends on the plant species. While the role of asparagine in germination is relatively well documented, data on its participation in senescence are quite scarce and are contradictory. On one side, post-harvest or darkinduced senescence is characterized by an intense synthesis of asparagine (King et al., 1990; Nozawa et al., 1999; Lin and Wu, 2004), although asparagine also accumulates in plant tissues that are stressed or dark-adapted (Urquart and Joy, 1981; Rabe, 1990). On the other side, only traces of asparagine are found in the natural senescence of radish cotyledons (Nozawa et al., 1999). Yet, in a previous study, King et al. (1995) reported the induction of an asparagine synthetase gene in asparagus leaves under natural senescence, although asparagine content of senescing leaves was not determined. Sunflower is an important crop plant whose nitrogen metabolism is under-researched compared to other model plants. To date, the role of asparagine in sunflower germination and senescence remains undetermined. In plants, asparagine is synthesized by asparagine synthetase (AS; EC 6.3.5.4.). Sunflower possesses two class I (HAS1, HAS1.1) and one class II (HAS2) AS genes, and it is the plant in which most active AS genes have been identified (Herrera-Rodrı´guez et al., 2002). AS gene expression has been linked to both asparagine synthesis and accumulation in plant tissues (Lam et al., 1998; Herrera-Rodrı´guez et al., 2004). Class I and class II AS genes have been shown to be regulated by sucrose, glutamine, asparagine and other amino acids (Lam et al., 1998; HerreraRodrı´guez et al., 2002, 2004). However, neither the extent of their contribution to the synthesis of
M.B. Herrera-Rodrı´guez et al. asparagine for germination and natural senescence nor the regulation of their expression in these events is well established. Sunflower AS genes are differentially regulated, showing distinct expression patterns (Herrera-Rodrı´guez et al., 2004). Consequently, they may play different roles in varying situations which require asparagine for nitrogen mobilization. Sunflower is a plant with epigeal germination, whose cotyledons, after germination, expand and turn into photosynthetic organs that finally undergo senescence. Sunflower cotyledons can be a suitable model to study the two main nitrogen mobilization processes and to compare leaf and cotyledon senescence. To investigate asparagine and AS genes involvement in the main nitrogen mobilization processes in sunflower, the expression of HAS1, HAS1.1 and HAS2 genes, as well as the synthesis of asparagine and other nitrogen and carbon metabolites were studied during germination and natural senescence of cotyledons and leaves. The data revealed a role for asparagine in each mobilization situation, where it participates to differing degrees in comparison to glutamine. Different combinations of AS genes support asparagine synthesis for germination or for senescence.
Materials and methods Plant culture conditions and sample collection Sunflower plants (Helianthus annuus L.) from the isogenic cultivar HA-89 (Semillas Cargill SA, Sevilla, Spain) were grown under a 16-h photoperiod with irradiance of 200 mmol m2 s1 PAR (provided by Sylvania cool white F72T12/CW/VHO, 160 W fluorescent lamps supplemented with Mazda 60 W incandescent bulbs) and day/night temperature and relative humidity regimes of 23 1C/19 1C and 70%/80%, respectively. Seeds were imbibed in water for 3 h and placed in plastic trays, containing wetted cellulose paper until germination was completed (5 days). Seedlings showing a similar degree of development were transferred to plastic trays containing a 2:1 (v:v) mixture of perlite and vermiculite. Seedlings were irrigated daily with standard nutrient solution containing 10 mM KNO3 (Hewitt, 1966), and were cultured in trays for up to 31 days. For longer culture periods, plants were transferred individually to pots containing Composana substrate (Compo GmbH, Germany). All plant samples were collected at the same time of the day
ARTICLE IN PRESS Asparagine synthetase genes in sunflower germination and senescence and were frozen by immersion in liquid nitrogen immediately after collection and stored at 80 1C until use.
Analysis of RNA Total RNA was isolated from different organs using the TRI REAGENTs – RNA/DNA/Protein Isolation Reagent (Molecular Research Center, Inc). Gene-specific probes for HAS1, HAS1.1 and HAS2 were prepared from their 30 untranslated regions as previously described (Herrera-Rodrı´guez et al., 2002). Samples of total RNA from different organs were separated by denaturing electrophoresis in formaldehyde agarose gels and blotted onto nylon filters according to standard procedures (Sambrook et al., 1989). Hybridization was performed at 42 1C, overnight, in solutions containing 50% (v:v) formamide and the corresponding gene-specific probe. After hybridization, filters were washed twice for 15 min in 0.2 SSC, 0.1% (w:v) SDS at 65 1C. Results of Northern analyses were revealed by autoradiography after exposing X-ray films (Kodak X- Omat AR) to the filters for 15 days at 80 1C.
1063
Spherisorb ODS-2 HPLC column (150 4 mm, 5 mm) and the dansyl-derivatives (ammonium and amino acids) separated at 30 1C using a mobile phase consisted of two eluents: 0.5% (v:v) acetic acid and 0.0075% (v:v) triethylamine in water (eluent A) and pure acetonitrile (eluent B). The gradient program was as follows: 17.5% eluent B for 10 min (flow rate at 1 mL min1), 17.5–35% eluent B for 30 min (flow rate from 1 to 1.5 mL min1), and 35–55% eluent B for 20 min (flow rate from 1.5 to 2 mL min1). Asparagine, glutamine and ammonium concentrations were calculated by measures at 254 nm using the corresponding standards. Leaf and cotyledon chlorophyll concentrations were determined spectrophotometrically at 652 nm as described by Arnon (1949). All analytical determinations were carried out on extracts from several separate plants harvested randomly, and were measured in triplicate. The data are mean values7SD. Experiments were replicated three times with very similar results.
Results
Analysis of metabolites
Amino acids, ammonium and sucrose content during cotyledon development
Frozen powdered leaf and cotyledon materials (0.10–0.15 g) were extracted for 30 min at 80 1C in screw-cap tubes as follows: twice with 1 mL of 80% aqueous ethanol (buffered with 5 mM Hepes-KOH, pH 7.5), once with 1 mL of 50% aqueous ethanol (buffered as before) and once with 1 mL of distilled water. The extract was centrifuged between each step (14,000g, 5 min), and the four supernatants collected, combined and kept at 20 1C until carbohydrate determinations. Sucrose in the extracts was measured as described in Stitt et al. (1989). Total free amino acids were determined in the same extract with ninhydrin by the cadmiumninhydrin method, modified from Dreyer and Bynum (1967). For asparagine, glutamine and ammonium determination, frozen powdered plant material (0.2 g) was extracted twice with 1 mL of 50% aqueous ethanol for 30 min at 4 1C. The extract was centrifuged between each step (13,000g, 5 min), and the two supernatants collected were combined. Subsequently, this supernatant (1 mL) was dansylated by adding 250 mL of 1% (w:v) Na2CO3 and 250 mL dansyl chloride (10 mg mL1 acetonitrile). Samples were vortexed gently and incubated in the dark for 30 min at 60 1C. Following the incubation, the dansylated extract (20 mL) was injected into a
Sunflower cotyledons are very remarkable organs, playing three different nutritional roles in seedling development. First, as nutrient storage organs, they feed the root axis growth during germination. Second, as the stored reserves are exhausted, cotyledons undergo a greening and expanding process that transforms them into the first organs providing photosynthetic resources for the seedling (Fig. 1). Sunflower cotyledons were visibly green by day 6, and reached their maximum chlorophyll content by day 10 (Fig. 2). By day 16, cotyledons had expanded to 85% of their final size. Cotyledons, in their third nutritional role, enter a senescence process that returns, to the plantlet, a portion of the materials invested in their construction. Chlorophyll loss indicating the start of cotyledon senescence was observed at day 13. By day 31, cotyledons were entirely yellow and showed necrotic edges (Figs. 1 and 2). Glutamine and asparagine are relevant N-transport compounds in plants. The asparagine and glutamine contents in sunflower cotyledons were followed from germination to senescence to assess their contribution to the nitrogen management in the different stages of cotyledon and seedling development (Fig. 4A). Dry seed contained much less glutamine than asparagine, which was the
ARTICLE IN PRESS 1064
M.B. Herrera-Rodrı´guez et al.
Figure 1. Sunflower cotyledon development. (a) Dry seed, (b) dry seed cotyledons, (c) 3-day-old seedling, (d) 10-dayold plantlet and cotyledon (inset), (e) 22-day-old plant and cotyledon (inset), (f) 31-day-old plant and cotyledon (inset).
Figure 2. Chlorophyll content during leaf and cotyledon development. Cotyledon (K) and primary leaves (’) were harvested from sunflower plants and their chlorophyll content determined. Plant’s age in days-aftersowing is indicated on the x-axis. Each value is the mean7SD of the results from three separate determinations.
predominant amide during germination. By the end of germination (days 3–4), both glutamine and asparagine contents were greatly increased. Glutamine levels were three times those of asparagine at the beginning of the expansion period. Total
amino acid content also showed a sharp increase around the beginning of the cotyledon expansion period (Fig. 4B). Six days after the initiation of the expansion (day 10; cotyledon at 53% of their final size), the concentrations of both amides, as well as total amino acids, had decreased to minimum values, and remained low and relatively stable throughout senescence. Sucrose has been reported to regulate sunflower AS genes (Herrera-Rodrı´guez et al., 2004). The evolution of sucrose content during cotyledon development could help to explain the observed AS gene expression patterns. Sucrose content in cotyledons from the dry seed and during the early steps of germination was well above that found in illuminated mature leaves (Figs. 4B and 5B). Preceding glutamine and asparagine accumulation, sucrose content dramatically increased from the middle of germination (day 2), to the beginning of expansion (day 6). Sucrose levels were strongly reduced soon before and during senescence (Fig. 4B). All three sunflower AS genes have been reported to be induced by ammonium accumulation
ARTICLE IN PRESS Asparagine synthetase genes in sunflower germination and senescence (Herrera-Rodrı´guez et al., 2004). Ammonium in cotyledons was low until the end of germination, but increased by the expansion period, (maximum value about 5 mmol g1 FW), and finally returned to low values at and following day 10 (Fig. 4A).
Expression of HAS1, HAS1.1 and HAS2 during cotyledon development To determine which sunflower AS gene or genes were participating in germination, expansion and senescence, the relative abundance of HAS1, HAS1.1 and HAS2 transcripts was determined in 15 successive stages of the cotyledon development (Fig 3). Dry sunflower seeds contained no detectable AS mRNA, which had to be transcribed de novo after seed imbibition. HAS2 transcripts were detected as early as on day 1, and strongly accumulated in the cotyledons for the next 2 days. According to the mRNA level, HAS2 expression declined by the end of germination (Fig. 3, day 4), and remained almost inactive until senescence, when a very slight accumulation of its transcripts was noted (Fig. 3, days 22 and 25).
1065
HAS1 expression was restricted to days 1 and 8, while no HAS1.1 mRNA was detected during any of the 15 developmental stages tested.
Amino acids, ammonium and sucrose in sunflower leaf senescence Leaf senescence causes a thorough mobilization of nitrogen in which asparagine and, hence, AS genes may play a role. Sunflower primary leaves reached their maximum size (about 9 cm length) by day 20, and their maximum chlorophyll content by day 23 (Fig. 2). Chlorophyll loss detected by day 27 indicated that senescence had started. Thirty-six and 40-day primary leaves, retaining 50% and 24% of their maximum chlorophyll content, respectively, were considered as fully senescent (Fig. 2). Both asparagine and glutamine contents increased in sunflower leaves by late senescence (Fig. 5A, day 40), while total amino acid content was slightly reduced (Fig. 5B). Ammonium content was also reduced by leaf senescence, as it happened in cotyledon senescence (Figs. 4A and 5A). Sucrose content of primary leaves was reduced by the end of their development. Early senescent leaves still retained 90% of the young leaves sucrose content. In late senescent leaves (36- and 40-day leaves), sucrose content was decreased by 50%, which may have affected the expression of sucroserepressed genes (Fig. 5B).
Expression of HAS1, HAS1.1 and HAS2 during leaf senescence
Figure 3. HAS1, HAS1.1 and HAS2 expression during sunflower cotyledon development. Cotyledons were sampled from sunflower plants on the days after sowing specified on the figure. Twenty micrograms of total RNA from the sampled cotyledons were electrophoresed, stained with ethidium bromide (rRNA) and blotted onto a nylon filter. The filter was hybridized sequentially to probes specific for HAS1, HAS1.1 and HAS2. The old probe was stripped off the filter before each new hybridization. Results of hybridization were revealed by autoradiography after 15 days of exposure.
To identify the AS genes that contribute to the synthesis of asparagine for sunflower natural leaf senescence, the HAS1, HAS1.1 and HAS2 relative transcript abundance was determined in mature (20-day), early (27-day) and late senescent (36- and 40-day) leaves. According to their transcript levels, HAS1 and HAS1.1 were expressed only during leaf senescence, with their expression restricted to the latest stages. On the other hand, the HAS2 expression level observed in mature leaves remained unchanged after the onset of senescence (Fig. 6).
Discussion Asparagine is the prevailing amide throughout sunflower germination (Fig. 4A). Its increased content in the transition from germination to expansion indicates that asparagine also makes a considerable contribution to the transformation of
ARTICLE IN PRESS 1066
M.B. Herrera-Rodrı´guez et al.
Figure 4. Sucrose, ammonium and amino acids content during sunflower cotyledon development. Cotyledons obtained from the plants used in the experiment depicted in Fig. 3 were analyzed for asparagine, glutamine, ammonium (A), sucrose and total amino acids (B). Cotyledon’s age in days-after-sowing is indicated on the x-axis. Each value is the mean7SD of the results from three separate determinations.
cotyledons into photosynthetic organs. The early HAS2 expression in germination is likely responsible for the increased asparagine synthesis at the beginning of the expansion period. Herrera-Rodrıguez et al. (2004) previously found a direct relationship between AS transcript level and asparagine accumulation in sunflower tissues. Kern and Chrispeels (1978) reported an AS activity peak on the third day of germination in V. radiata cotyledons, which is consistent with the early AS gene expression in sunflower. AS gene expression has been observed to begin later (days 8–10) in pea cotyledons (Tsai and Coruzzi, 1990). Senescence, like germination, is regarded as a nitrogen mobilization period, and asparagine as a major compound for nitrogen translocation. However, the reduced asparagine content and the very
low AS gene expression suggest that asparagine is not making a substantial contribution to nitrogen mobilization during natural senescence of sunflower cotyledons. Preliminary results showed HAS1 expression in 20-day-old sunflower cotyledons (Herrera-Rodrıguez et al., 2002). However, in further detailed experiments, such as the one illustrated in Fig. 1, we were unable to detect any significant HAS1 induction in senescing cotyledons, in which asparagine content is dramatically reduced. These experiments have led us to believe that the preliminary results were most likely an artifact. In a low metabolite level context, asparagine prevails over glutamine during most parts of senescence. Asparagine content in senescing cotyledons was in the range (0.2–0.6 mmol g1 FW) of what occurs in senescent leaves (Figs. 4A and 5A).
ARTICLE IN PRESS Asparagine synthetase genes in sunflower germination and senescence
1067
Figure 5. Sucrose, ammonium and amino acid content during sunflower leaf development. Mature (20-day-old) or senescent (27, 36 and 40-day-old) leaves were analyzed for asparagine, glutamine, ammonium (A), sucrose and total amino acids (B). Each value is the mean7SD of the results from three separate analyses.
Leaf and cotyledon senescence are considered very similar processes. Indeed, the early cotyledon senescence has sometimes been proposed as a model for the study of leaf senescence. Leaf senescence in sunflower differs from cotyledon senescence in that all three sunflower AS genes are expressed throughout. These genes are regulated, in opposite ways, by carbon and nitrogen metabolites (Herrera-Rodrı´guez et al., 2004). HAS2 exhibits a rather constitutive expression which is enhanced by sucrose. On the contrary, HAS1 and HAS1.1 are repressed by sucrose. HAS1, HAS1.1 and HAS2 expression during leaf senescence is consistent with the metabolic regulation mentioned above, as the rather constitutive HAS2 is not repressed by the reduction in sucrose content, while HAS1 and HAS1.1 are not active until such reduction takes place. The same metabolic regulation is expected to prevail during cotyledon development. Dry seeds contain no AS gene transcripts that are detec-
ted as soon as metabolic activity is restarted (day 1). During the next days, an extraordinarily high level of sucrose is produced, and this may have contributed to the intense HAS2 induction on days 2 and 3. HAS2 transcript levels were reduced by day 4, still with elevated sucrose content, but coinciding with the highest accumulation of glutamine, asparagine and total free amino acids (Fig. 4). Repression by amino acids, particularly asparagine and glutamine, characterizes class II AS genes such as HAS2 (Lam et al., 1998; Herrera-Rodrı´guez et al., 2004). On day 6, when HAS2 transcripts became almost undetectable, the level of glutamine, asparagine and total free amino acids was still high, while sucrose content was lowered. By day 8, sucrose content was reduced to below the day 1 level, while total amino acids and, particularly, glutamine content was much higher, thus establishing a metabolic context unfavorable to HAS2 expression.
ARTICLE IN PRESS 1068
Figure 6. HAS1, HAS1.1 and HAS2 expression during sunflower leaf senescence. Leaves were sampled from the plants used in the experiment depicted in Fig. 5. Leaf’s age in days-after-sowing is indicated on the figure. Twenty micrograms of total RNA from leaf material were electrophoresed, stained with ethidium bromide (rRNA) and blotted onto a nylon filter. The filter was hybridized sequentially to probes specific for HAS1, HAS1.1 and HAS2. The old probe was stripped off the filter before each new hybridization. Results of hybridization were revealed by autoradiography after 15 days of exposure.
To this point, HAS2 expression pattern agrees with the proposed metabolic regulation by the C/N status controlling class II AS genes (Lam et al., 1998; Herrera-Rodrı´guez et al., 2004). However, the absence of a sustained HAS2 expression from day 10 forward cannot be easily explained in these terms. From days 10 to 16, sucrose and total amino acid contents of cotyledons (average 1.3 and 9.8 mmol g1 FW, respectively) were more favorable
M.B. Herrera-Rodrı´guez et al. to HAS2 expression than those occurring in leaf senescence at day 36 (1.1 mmol sucrose g1 FW and 19.2 mmol total amino acids g1 FW). Asparagine and glutamine contents are also lower than that in late leaf senescence, when HAS2 is allowed to express. Later, between days 20 and 31, sucrose content was further reduced (average 0.57 mmol g1 FW), which may have negatively affected HAS2 expression. However, HAS2 has been shown to resist sucrose reductions from 2.7 to 0.40 mmol g1 FW in leaves, without a significant reduction of transcript abundance (Herrera-Rodrıguez et al., 2004). AS gene regulation may be more complex than what it has been assumed, and the possibility of novel cotyledon-specific metabolic or non-metabolic elements repressing HAS2 during senescence cannot be discarded. Unfortunately, no data on class II AS gene regulation during natural senescence in other plants are currently available to compare to our results. HAS1 and HAS1.1 are class I AS genes whose expression is usually repressed in illuminated leaves but can be induced by carbon deficiency or nitrogen excess (Tsai and Coruzzi, 1991; Chevalier et al., 1996; Shi et al., 1997; Lam et al., 1998; Herrera-Rodrı´guez et al., 2002, 2004). Their induction during sunflower leaf senescence is associated to a sucrose decrease (from 2.0 to 1.1 mmol g1 FW) and agrees with the mentioned metabolic regulation. The same is true for their repression in cotyledon during germination and expansion, when sucrose content is well above 1.0 mmol g1 FW. However, on day 1 and especially on day 8, a moderate amount of sucrose combined with a fairly large amount of total free amino acids allowed HAS1 expression. However, no HAS1 or HAS1.1 expression was detected in cotyledon senescence, although sucrose content was lowered (0.57 mmol g1 FW average sucrose content on days 20–31) behind the level that allows HAS1 and HAS1.1 expression in leaf senescence (average 1.1 mmol g1 FW). Then, as in the case of HAS2, HAS1 and HAS1.1 were not expressed in cotyledon senescence even under what seemed a favorable metabolic context. High ammonium accumulation (about 8 mmol g1 FW), due to an exogenous supplement, up-regulated the expression of all three sunflower AS genes in roots (Herrera-Rodrı´guez et al., 2004). In germination and leaf senescence, AS gene expression takes place without great ammonium accumulation. In addition, the similar ammonium content of senescing leaf and cotyledons (average 0.5 and 0.6 mmol g1FW, respectively) does not help to explain the absence of AS gene expression in the latter.
ARTICLE IN PRESS Asparagine synthetase genes in sunflower germination and senescence Such a difference between cotyledon and leaf senescence has been reported in other species. ASN1, an Arabidopsis class I AS gene, is greatly induced in naturally senescing Arabidopsis leaves (Fujiki et al., 2001). However, Nozawa et al. (1999) detected no AS transcripts during natural senescence of radish cotyledons, in which is, to our knowledge, the only work available on the role of asparagine and AS genes in cotyledon senescence. All of these findings again show the existence of a cotyledon-specific down-regulation of AS genes during senescence, whose nature has not yet been established. Nozawa et al. (1999) suggested that the conversion of cotyledons from a sink to a source organ could trigger an aging-related regulation, preventing AS gene expression, but this seems less probable since in senescing leaves, in which such a conversion also takes place, AS gene expression is allowed. In summary, our results show that asparagine is involved in nitrogen management during sunflower germination, and also contributes to the growth and transformation of cotyledons into photosynthetic organs. Asparagine may also play a role in nitrogen mobilization in leaf and cotyledon natural senescence, although in a context of reduced metabolite level. Different sunflower AS genes are responsible for the synthesis of asparagine during these high mobilization situations. Early HAS2 expression provides most of the asparagine synthetase needed to produce asparagine for cotyledon and seedling growth. The asparagine synthetase required for mature leaf maintenance is also provided by HAS2, and that needed for leaf senescence is contributed by HAS2, reinforced by the induction of HAS1 and HAS1.1. In most of the cases, excepting cotyledon senescence, the activity of those AS genes seems to be regulated by the prevailing C/N status.
Acknowledgments This work was funded by grants from Direccio ´n General de Ensen ˜anza Superior e Investigacio ´n Cientı´fica (DGESIC, BXX 2000-0289 and BOS200301595) and by Plan Andaluz de Investigacio ´n (PAI, group CV-159), Spain. We thank Marga Pe´rezJime´nez for her help in HPLC determinations.
References Arnon DI. Copper enzymes in isolated chloroplast. Polyphenol oxidase in Beta vulgaris. Plant Physiol 1949;24:1–15.
1069
Capdevila AM, Dure L. Developmental biochemistry of cottonseed embryogenesis and germination. Plant Physiol 1977;59:268–73. Chevalier C, Bourgeois E, Just D, Raymond P. Metabolic regulation of asparagine synthetase gene expression in maize (Zea mays L.) root tips. Plant J 1996;9:1–11. Dreyer WJ, Bynum E. High-voltage paper electrophoresis. Methods Enzymol 1967;11:32–9. Fujiki Y, Yoshikawa Y, Sato T, Inada N, Ito M, Nishida I, et al. Dark-inducible genes from Arabidopsis thaliana are associated with leaf senescence and repressed by sugars. Physiol Plant 2001;111:345–52. Herrera-Rodrı´guez MB, Carrasco-Ballesteros S, Maldonado JM, Pineda M, Aguilar M, Pe´rez-Vicente R. Three genes showing distinct regulatory patterns encode the asparagine synthetase of sunflower (Helianthus annuus). New Phytol 2002;155:33–45. Herrera-Rodrı´guez MB, Maldonado JM, Pe´rez-Vicente R. Light and metabolic regulation of HAS1, HAS1.1 and HAS2, three asparagine synthetase genes in Helianthus annuus. Plant Physiol Biochem 2004;42: 511–8. Hewitt EJ. Sand and water culture methods used in the study of plant nutrition. Wallingford: CAB Internacional; 1966. Kern R, Chrispeels MJ. Influence of the axis on the enzymes of protein and amide metabolism in the cotyledons of mung bean seedlings. Plant Physiol 1978;62:815–9. King GA, Woollard DC, Irving DE, Borst WM. Physiological changes in asparagus spears tips after harvest. Physiol Plant 1990;80:393–400. King GA, Davies KM, Richard JS, Borst WM. Similarities in gene expression during the postharvest-induced senescence of spears and natural folisr senescence of asparagus. Plant Physiol 1995;108:125–8. Lam HM, Hsieh MH, Coruzzi GM. Reciprocal regulation of distinct asparagine synthetase genes by light and metabolites in Arabidopsis thaliana. Plant J 1998;16: 345–53. Larson LA, Beevers H. Amino acid metabolism in young pea seedlings. Plant Physiol 1965;40:424–32. Lea PJ, Miflin BJ. Transport and metabolism of asparagine and others nitrogen compounds within the plant. In: Miflin BJ, editor. The biochemistry of plants: amino acids and derivatives. New York, USA: Academic Press; 1980. p. 569–607. Lin JF, Wu SH. Molecular events in senescing Arabidopsis leaves. Plant J 2004;39(4):612–28. Nozawa A, Ito M, Hayashi H, Watanabe A. Dark-induced expression of genes for asparagine synthetase and cytosolic glutamine synthetase in radish cotyledons is dependent on the growth stage. Plant Cell Physiol 1999;40:942–8. Rabe E. Stress physiology: the functional significance of the accumulation of nitrogen-containing compounds. J Hortic Sci 1990;65:231–43. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1989.
ARTICLE IN PRESS 1070 Shi L, Twary SN, Yoshioka H, Gregerson RG, Miller SS, Samac DA, et al. Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves. Plant Cell 1997;9: 1339–56. Stitt M, Lilley RM, Gerhardt R, Heldt HW. Metabolite levels in specific cells and subcellular compartments of plant leaves. Methods Enzymol 1989;174: 518–52.
M.B. Herrera-Rodrı´guez et al. Tsai FY, Coruzzi GM. Dark-induced and organ-specific expression of two asparagine synthetase genes in Pisum sativum. EMBO J 1990;9:323–32. Tsai FY, Coruzzi GM. Light represses transcription of asparagine synthetase genes in photosynthetic and nonphotosynthetic organs of plants. Mol Cell Biol 1991;11:4966–72. Urquart AA, Joy KW. Use of phloem exudates technique in the study of amino acid transport in pea plant. Plant Physiol 1981;68:750–4.
