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Independent roles of methionine and O-acetyl-l-serinein the regulation of the β subunit gene of β-conglycininMasami Yokota Hirai a c , Hoyeun Kim a d , Hiroaki Hayashi a , Mitsuo Chino a e , Satoshi Naitob & Torn Fujiwara aa Department of Applied Biological Chemistry, Graduate School of Agricultural and LifeSciences , The University of Tokyo , Tokyo, 113-8657, Japanb Division of Applied Bioscience, Graduate School of Agriculture , Hokkaido University ,Sapporo, 060-8589, Japanc Faculty of Pharmaceutical Sciences , Chiba University , Chiba, 263-8522, Japand Department of Biology , Seoul National University , Seoul, 151-742, Koreae Department of Bio-Production Science, Faculty of Bioresource Science , Akita PrefecturalUniversity , Akita, 010-0146, JapanPublished online: 22 Nov 2011.

To cite this article: Masami Yokota Hirai , Hoyeun Kim , Hiroaki Hayashi , Mitsuo Chino , Satoshi Naito & Torn Fujiwara (2002)Independent roles of methionine and O-acetyl-l-serine in the regulation of the β subunit gene of β-conglycinin, Soil Scienceand Plant Nutrition, 48:1, 87-94, DOI: 10.1080/00380768.2002.10409175

To link to this article: http://dx.doi.org/10.1080/00380768.2002.10409175

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Soil Sci. Plant Nutr., 48 (1), 87 -94,2002

Independent Roles of Methionine and O-Acetyl-L-Serine in the Regulation of the J3 Subunit Gene of J3-Conglycinin

Masami Yokota Hirai I, Hoyeun Kim", Hiroaki Hayashi, Mitsuo Chino3,

Satoshi Naito*, and Torn Fujiwara

Department of Applied Biological Chemistry. Graduate School of Agricultural and Life Sciences, The Ulliversity of Tokyo. Tokyo, 113 - 8657 Japan; and *Divisioll of Applied Bioscience, Graduate School of Agriculture, Hokkaido

University, SappolV, 060- 8589 Japan

Received August 14. 2001; accepted in revised form October 19, 2001

The gene encoding the p subunit of p-conglycinin, one of the major seed storage proteins of soybean (Glycine max [L.] Merr.) , is downregulated by methionine (Met) and upregulated by O-acetyl-L-serine (OAS). We examined the interaction between Met and OAS in the regu­lation of the expression of the P subunit gene in in vitro-cultured immature soybean cotyle­dons. Application of Met reduced the accumulation of the p subunit both in the presence and absence of OAS in the media: Application of OAS increased the accumulation of the p subunit both in the presence and absence of Met. We also studied the regulation of the p subunit gene promoter by sulfur in Arabidopsis thaliana (L.) Heynh. mutant mtol-l, that over-accumulates soluble Met, using the p-glucuronidase (GUS) gene as a reporter. When grown under sulfur-deficient conditions, the GUS activity in seeds of the transgenic plants carrying the mtol-l mutation was lower than that of the plants which do not carry the mutation, suggesting that the p subunit gene promoter was downregulated by the mtol-l mutation under sulfur deficiency. When the transgenic plants carrying the mtol-l mutation were grown under control and sulfur-deficient conditions, the GUS activity was higher under sulfur-deficient conditions, suggesting that the p subunit gene promoter was upregu­lated by sulfur deficiency even in the mtol-l mutant background. Furthermore, OAS con­tents in immature siliques were elevated both in the mtol-l mutant and wild-type plants under sulfur deficiency. Met contents in immature siliques of the mtol-l mutant plants increased even under sulfur deficiency. There was a strong positive correlation between the GUS activities in seeds and the endogenous [OAS] / [Met] ratio. These results suggest that OAS and Met regulate the p subunit accumulation through independent pathways.

Key Words: Arabidopsis thaliana, methionine, O-acetyl-L-serine, seed storage protein, soybean.

87

Accumulation of seed storage reserves varies depend­ing on the nutritional conditions of the mother plant. The response of seed storage protein accumulation to sulfur nutrition has been reported in a number of plant species (Hawkesford 2000; Saito 2000). When plants are cultured under sulfur deficiency, accumulation of sulfur-rich proteins is reduced while that of sulfur-poor proteins is enhanced. This response may be considered to be an adaptive reaction of plants for the accumulation

of a large amount of protein, and thus nitrogen, in seeds under sulfur-limited conditions.

Present addresses: 1 Faculty of Pharmaceutical Sciences. Chiba University, Chiba, 263 - 8522 Japan. 2 Department of Biology. Seoul National University, Seoul, 151- 742 Korea. 3 Depart­ment of Bio-Production Science, Faculty of Bioresource Sci­ence, Akita Prefectural University, Akita, 010-0146 Japan. The first two authors contributed equally to this work.

In soybean (Glycine max [L.] Merr.), one of the two major seed storage proteins, glycinin (lIS globulin), is downregulated under sulfur deficiency while that of the other, ~-conglycinin (7S globulin), is upregulated (Gay­ler and Sykes 1985). ~-Conglycinin is composed of three subunits, namely a, a', and ~. Among these sub­units, the ~ subunit has the lowest content of sulfur-con­taining amino acids (Coates et al. 1985) and the response of the accumulation of the ~ subunit to sulfur deficiency is most pronounced (Gayler & Sykes 1985). Upregulation of the ~ subunit accumulation under sulfur deficiency can be demonstrated in transgenic petunia and Arabidopsis thaliana plants and the response is reg-

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88 M.Y. HIRAI et al.

ulated mainly at the level of transcription (Fujiwara et al. 1992; Naito et al. 1994a; Hirai et al. 1995).

We have recently identified O-acetyl-L-serine (OAS) as a key metabolite involved in the upregulation of the r3 subunit gene expression under sulfur deficiency (Kim et al. 1999). OAS accumulates under sulfur deficiency both in soybean and in A. tlzaliana. Application of OAS increased the levels of the r3 subunit protein and mRNA in soybean immature cotyledon cultures, in a way simi­lar to sulfur deficiency (Kim et al. 1999), although nor­mal levels of sulfate were present in the media. OAS is a precursor of cysteine biosynthesis, and sulfur and nitro­gen assimilation pathways merge at this step. Therefore, it is reasonable to assume that the OAS contents are upregulated under a low sulfur / nitrogen ratio and downregulated under a high sulfur / nitrogen ratio. In fact, changes in sulfur and nitrogen nutrition exert a par­allel effect on the levels of OAS and the expression of the r3 subunit gene (Hirai et al. 1997; Kim et al. 1999).

OAS is also known to regulate sulfur assimilation and sulfur nutrition-regulated genes. Accumulation of mRNA for a sulfate transporter gene in wheat has been shown to increase following the exogenous application of OAS (Smith et al. 1997). A sulfate transporter gene of A. thaliana was also upregulated in roots upon the appli­cation of OAS (Hatzfeld and Saito 2000). Accumulation of mRNA for adenosine 5' phospho sulfate reductase is downregulated by nitrogen starvation and is restored by the application of OAS in A. thaliana roots (Koprivova et al. 2000). Neuenschwander et al. (1991) have also demonstrated that OAS is involved in the light regula­tion of sulfate assimilation in Lemlla.

Methionine (Met) is another key metabolite that affects the expression of the r3 subunit gene of r3-congly­cinin. Accumulation of the r3 subunit protein was re­pressed to a very low level when Met was applied to the in vitro culture system of soybean immature cotyledons (Holowach et al. 1984a). This response has been found to be regulated at the level of transcription (Creason et al. 1983; Holowach et al. 1986). In transgenic A. thaliana, the effect of Met on the r3 subunit gene pro­moter was evidenced by the exogenous application of Met in detached immature silique cultures and in the whole plant (Hirai et al. 1994), suggesting that the r3 subunit gene promoter responds to Met in A. tlzaliana.

The mto 1-1 mutation of A. thaliana leads to an over­accumulation of soluble Met (Inaba et aI. 1994). Met is synthesized from aspartic acid along with lysine, threo­nine, and isoleucine (Matthews 1999, for review). Cys­tathionine ,),-synthase (CGS) catalyzes the first com­mitted step in Met biosynthesis which is considered to be a key regulatory step in Met biosynthesis. We have recently observed that the accumulation level of CGS mRNA is subject to a negative feedback regulation at

the step of mRNA stability and that the I11t01-1 mutation abolishes this regulation (Chiba et al. 1999). The mt01 mutations are clustered in a coding region of about 80 amino acid residues from the N-terminus of CGS, and the amino acid sequence in the first ex on polypeptide (amino acids 1-183) is implicated in the regulation (Chiba et al. 1999). When the r3 subunit gene promoter­r3-glucuronidase (GUS) fusion gene was introduced into the mt01-1 mutant of A. thaliana through genetic cross­es, GUS activity in mature seeds decreased compared to that in the wild-type background (Naito et al. 1994b), indicating that the effect of Met may be studied by using the mt01-1 mutant.

Irrespective of the importance of OAS and Met in the regulation of the sulfur assimilation pathways, their interaction in the regulation of gene expression is not well understood. Holowach et al. (1984b) have reported the effect of Met and sulfatc concentrations on the r3 subunit protein accumulation in an ill vitro cotyledon culture of immature soybean seed. However, the effects of Met and OAS in vivo have not been reporeted in the literature. One may consider that the response of the r3 subunit gene of r3-conglycinin to sulfur deficiency and Met may involve the forward and reverse reactions, respectively, of the same regulatory system. However, the level of soluble Met is strictly regulated irrespective of sulfur availability. In fact, studies with Lemna have revealed that despite a 3000-fold difference in the avail­ability of sulfate in the media, the intracellular level of soluble Met did not change appreciably (Datko et al. 1978). Therefore, it remains to be determined whether the reactions to Met and sulfur deficiency, and thus to OAS, are independent or not.

Here, we studied the interactions of Met and OAS in the regulation of soybean seed storage protein genes. We used in vitro cultures of soybean immature cotyledons (Thompson et al. 1977) and hydroponic cultures of the mt01-1 mutant of A. thaliana. In vitro culture of imma­ture soybean cotyledons is a suitable system for the study of the nutrient control of seed storage protein accumulation (Thompson et al. 1977; Holowach et al. 1984a). In parallel, we cultured transgenic A. thaliana carrying the r3 subunit gene promoter-GUS fusion gene and the mt01-1 mutation under different conditions of sulfur nutrition and studied the expression of the r3 sub­unit gene promoter in relation to the accumulation of the metabolites. The data suggested that the effects of Met and OAS on the expression of the r3 subunit gene are exerted at least in part through independent pathways.

MATERIALS AND METHODS

Plant materials and culture conditions. Soy-

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Met and GAS Regulation of the ~ Subunit Gene 89

bean (Glycine max [L.] Merr. var. Toyosuzu) plants were grown in a greenhouse at 30'C day / 2YC night under natural light using a 2 : 1 mixture of vermiculite and perlite, and were watered with a 2000-fold dilution of a commercial liquid fertilizer (Hyponex, N-P-K = 8-12-6, Hyponex Corporation, Marysville, Ohio, USA) as described previously (Kim et al. 1999). In vitro culture of immature soybean cotyledons was performed accord­ing to the method of Thompson et al. (1977). In brief, pods were harvested at about three weeks after flower­ing when embryos were about 30-50 mg in fresh weight. The f3 subunit protein was not detectable at this stage. The pods were surface-sterilized, the embryos taken out, and the embryonic axis removed. One cotyle­don from an embryo was placed in 5 rnL of basal medi­um (Thompson et al. 1977) in a 30 rnL flask, while the other cotyledon was placed in a medium supplemented with different concentrations of Met and / or ~AS. The cotyledons were cultured for six days at 25'C with gen­tle agitation under fluorescent lamps. After the culture, cotyledons were blot-dried, weighed, frozen immediate­ly with liquid N 2, and stored at -80'C until use.

Transgenic A. thaliana (L.) Heynh. strains SNTf33 and SNTf33mto were described previously (Naito et al. 1994b). Both strains carry the GUS gene under the con­trol of the f3 subunit gene promoter. SNTf33mto also car­ries the mt01-1 mutation (Inaba et al. 1994; Naito et al. 1994b; Chiba et al. 1999). Arabic/apsis plants were grown hydroponically at 22'C under natural light as described previously (Hirai et al. 1995). The culture media contained l.5 mM (control media) or 30 fLM sul­fate (sulfate-deficient media), as described previously (Hirai et al. 1995).

Extraction of seed storage protein from soy­bean and SDS-PAGE. Proteins were extracted from soybean cotyledons after 6 d of culture, as described previously (Kim et al. 1999). Protein concentrations were determined using Protein Assay (BioRad, Her­cules, CA USA) with bovine serum albumin as a stan­dard (Bradford 1976). Thirty mg of proteins were separated by SDS-PAGE in 12.5% polyacrylamide slab gels (Laemmli 1970), and stained with Coomassie Bril­liant Blue R-250.

Measurement of OAS and Met concentra­tions. The concentrations of Met and OAS were mea­sured as described previously (Kim et al. 1997, 1999). In short, the fifth to seventh siliques on the primary inflorescence of transgenic A. thaliana strains SNTf33 and SNTf33mto were harvested at 10 d after flowering (DAF). Amino acids were extracted with 10 mL of 80% ethanol per gram tissue at 45'C. After centrifugation, the supernatants were collected and the solvents evaporated to dryness in vacuo at room temperature. The residues were then dissolved in appropriate amounts of 0.01

N HCI and subjected to HPLC analysis (model L-6200 Hitachi. Tok.)'o, Japan) using a No. 2619 column (Hi­tachi) at 3TC. Met and OAS were eluted by 24 mM trilithium citrate pH 2.77, 35 mM lithium chloride, 113 mM citric acid and 2.8% ethanol, and detected with a fluorometer after reaction with o-phthalaldehyde.

Determination of GUS activity. Mature seeds were harvested from the fifth to seventh siliques on the primary inflorescence of transgenic A. tlzaliana strains SNTf33 and SNTf33mto and subjected to flu oro metric GUS assays. GUS activity was measured as described previously (Hirai et al. 1994), using 4-methylumbellifer­yl-f3-D-glucuronide as a substrate (Jefferson et al. 1987). The amount of total protein was measured using Protein Assay (BioRad, Hercules, CA, USA).

RESULTS

Effects of OAS and Met on soybean seed storage protein accumulation in immature co­tyledon cultures

Accumulation of soybean seed storage proteins was studied in the in vitro culture of soybean immature coty­ledons. Accumulation of the f3 subunit protein of f3-con­glycinin was repressed by exogenous application of 2 mM Met (Fig. 1, lanes 3 and 7; Holowach et al. 1986) and enhanced by exogenous application of 1 or 10 mM OAS (Fig. I, lanes 2 and 6: Kim et al. 1999). On the other hand, accumulation of glycinins was reversely affected by each treatment (Fig. 1; Holowach et al. 1986; Kim et al. 1999).

To determine whether or not either of the effects is dominant over the other in terms of regulation of the seed storage protein accumulation, we applied Met and OAS simultaneously to the in vitro culture system. The pattern of seed storage protein accumulation (both the f3 subunit and glycinins) in the medium containing 10 mM

OAS and 2 mM Met (Fig. 1, lane 4) was intermediate between that in the media containing 10 mM OAS with­out Met (lane 2) and 2 mM Met without OAS (lanes 3 and 7), and similar to that in the control (lanes 1 and 5).

Application of 1 mM OAS to 2 mM Met medium did not alter appreciably the pattern of seed storage protein accumulation (compare lanes 7 and 8 in Fig. 1), al­though the application of 1 mM OAS alone enhanced the accumulation of the f3 subunit protein, compared with the control treatment (lanes 5 and 6).

These results suggest that the effects of OAS and Met are primarily additive, although the effect of one may become more prevalent than that of the other at low con­centrations.

OAS and Met concentrations in immature si-

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liques of A. thaliana mtol-l mutant plants cultured under sulfur deficiency

To determine whether Met and OAS affect the biosyn­thesis and / or metabolism of the other, we cultured the transgenic A. thaliana strain SNTr33mto carrying both the mto1-1 mutation and the r3 subunit promoter fused to the GUS reporter gene under sulfur deficiency. The

234 5 678

mto 1-1 mutation resulted in the over-accumulation of soluble Met in A. thaliana (Inaba et al. 1994), while sul­fate deficiency led to the accumulation of OAS in the siliques of A. thaliana (Kim et al. 1999).

SNTr33 and SNTr33mto plants were grown hydroponi­cally under control (1.5 mM sulfate) or sulfate-deficient (30 fLM sulfate) conditions and the concentration of

. a'] ' a ~-conglycinin

Fig. 1. Effects of Met and OAS application on seed storage protein accumulation in developing soy­bean cotyledons. Immature cotyledons of soybean were cultured ill vitro in the presence of Met and / or ~AS. After the culture, proteins were extracted and separated by SDS-PAGE followed by staining with Coomassie Brilliant Blue R-250. An equal amount of total proteins was loaded on each lane. Concentra­tions of OAS and Met, respectively, for each lane were as follows: Lane 1. 0 and 0 mM; Lane 2, 10 and o mM; Lane 3, 0 and 2 mM; Lane 4, 10 and 2 mM; Lane 5, 0 and 0 mM; Lane 6, 1 and 0 mM; Lane 7, 0 and 2. mM; Lane 8, 1 and 2 mM. Positions of the a', a, and [3 subunits of [3-conglycinin and acidic and basic subunits of glycinins are shown.

.-~

glycinin

20~---------------------------'

[ 01

:::::: a E c: ..... 10 * c:

* 2 c: a u en « 0

c ~s c ~s

SNT~3 SNT~3mto Fig. 2. OAS content in immature siliques of transgenic A. thaliana. Transgenic A. thaliana strains SNT[33 and SNT[33mto were cultured hydroponically at 0.03 (6S) or 1.5 mM (C) of sul­fate. OAS contents of the fifth to seventh siliques on the pri­mary inflorescence were measured at 10 DAF. The average ± SE of 4 to 5 replicates are shown. Means denoted by the same asterisk do not differ significantly at p < 0.1.

80~--------------------------'

§' 60 L1..

01 --a * E c: -..... c: 2 c: 40 a u Q) c: c: a i: +-' 20 Q)

~

o c ~s c ~s

SNT~3 SNT~3mto Fig. 3. Met content in immature siliques of transgenic A. thalia!![/. Transgenic A. thaliana strains SNT[33 and SNT[33mto were cultured hydroponically at 0.03 (6S) or 1.5 mM (C) of sul­fate. Met contents of the fifth to seventh siliques on the primary inflorescence were measured at 10 DAF. The average ± SE of 4 to 5 replicates are shown. Means denoted by the same asterisk do not differ significantly at p < 0.1.

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Met and OAS Regulation of the ~ Subunit Gene 91

GAS in the immature siliques at 10 DAF was measured. At this stage of seed development, seed storage protein genes including the ~ subunit gene are actively tran­scribed (Heath et al. 1986; Hirai et al. 1995). Under sul­fate deficiency, the concentration of GAS in SNT~3 increased 1.9-fold (Fig. 2; Kim et al. 1999). A similar increase in GAS concentration under sulfate deficiency was observed in SNT~3mto (Fig. 2). GAS concentration in SNT~3mto under the control condition was 9.1 runol per gram FW, and was 1.7-fold lower than that under sulfur deficiency. No significant difference was observed by the Student's t-test between the concentrations of GAS in SNT~3 and SNT~3mto under either control or sulfur-deficient conditions (Fig. 2). These results sug­gest that the free GAS concentration in siliques at 10 DAF was not affected by the mto1-1 mutation; that is, over-accumulation of soluble Met did not affect the bio­synthesis and metabolism of GAS.

The concentrations of Met in the immature siliques of SNT~3 and SNT~3mto at 10 DAF were also deter­mined. Under the control condition, the concentration in SNT~3mto was 1.3-fold higher than that in SNT~3

-c:: Q)

a .... a. C)

E --c:: E

::::: a E c:: -

c ilS c ilS

SNTP3 SNT~3mto

Fig. 4. GUS activity in mature seeds of transgenic A. tha/i­ana. Transgenic A. tha/ianG strains SNT~3 and SNT~3mto were cultured hydroponically at 0.03 (~S) or l.5 tel mM of sul­fate. GUS activity in mature seeds harvested from the fifth to seventh siliques on the primary inflorescence was measured. The average ± SE of 10 replicates is shown. Means were com­pared by Student's t-test. All means differed significantly at p

< 0.05.

(Fig. 3). The difference was significant. based on the Student's t-test (p < 0.05). Under sulfate deficiency, Met concentrations in SNT~3 and SNT~3mto decreased 0.7-fold (Fig. 3). It is notable that the mto1-1 mutation led to a 1.3-fold increase in Met concentration even under sulfate deficiency as well as under control condi­tions (Fig. 3). These results indicate that the mto 1-1 mutation exerts a major impact on the free Met concen­tration in the siliques of A. thaliana.

Response of the 13 subunit gene promoter to sulfur deficiency inA. thaliana mtol-l mutant

Mature seeds were harvested from SNT~3 and SNT­~3mto under control and sulfur-deficient conditions, and the GUS activity was measured. GUS activity in SNT~3 increased 7.9-fold under sulfate deficiency (Fig. 4; Hirai et al. 1995) and decreased 2A-fold in the mto1-1 mutant background (Fig. 4; Naito et al. 1994b). GUS activity in SNT~3mto cultured under sulfate deficiency was inter­mediate between that in SNT~3mto cultured under the control condition and SNT~3 cultured under sulfate deficiency (Fig. 4).

These results indicate that Met and sulfur deficiency (GAS), exert additive and independent effects on the ~ subunit gene expression in transgenic A. thaliana and that the regulation occurred mainly at the level of tran­scription. In other words, the methionine effect is exert­ed through a pathway that is at least in part distinct from that of sulfur deficiency .

DISCUSSION

In the present study, we demonstrated that: I) the effects of Met and GAS on the accumulation of soybean seed storage proteins were evident irrespective of the presence of the other; 2) the degree of induction of the GUS activity by sulfate deficiency in the mto1-1 mutant plants was similar to that of the wild-type plants; 3) downregulation of the GUS activity by the mto1-1 muta­tion was evident under sulfur deficiency; and 4) GAS accumulated at a higher concentration in the siliques of the mto 1-1 mutant plants under sulfate deficiency. These findings suggest the independent nature of the effects of Met and GAS in the regulation of the expression the ~ subunit gene.

We previously reported that the expression of the ~ subunit gene is upregulated by GAS in soybean cotyle­dons (Kim et al. 1999). We also demonstrated that GAS application did not exert a strong impact on the Met contents in soybean cotyledons cultured in vitro (Kim et al. 1999), suggesting that the effect of GAS is not medi­ated by a change in Met content. Simultaneous applica­tion of Met and GAS in this study confirmed the

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92 M.Y. HIRAI et al.

assumption that OAS and Met regulate the f3 subunit accumulation through independent processes. It is worth noting that independent regulation by Met and OAS also occurred in the accumulation of glycinin, another major component of seed storage proteins in soybean, at least at the level of protein accumulation, suggesting that such regulation is common to major seed storage pro­teins.

Induction of the f3 subunit gene promoter in the trans­genic A. thaliana strain SNTf33mto by sulfur deficiency indicated that the upregulation of the expression of the f3 subunit gene in response to sulfur deficiency occurred independently of the I1ltoJ-J mutation. It was also dem­onstrated that the mto1-1 mutation upregulates Met accumulation and downregulates the f3 subunit promoter under sulfur deficiency. These findings coincide with those obtained using the soybean cotyledon cultures de­scribed above.

The present results also suggest that the downregula­tion of the expression of the f3 subunit gene by Met does not occur through a change of OAS content. The level of OAS accumulation was not affected by the mto 1-J mutation, whereas the GUS activity was significantly decreased by the mto1-1 mutation, further suppOlting the independent nature of the regulation by Met and ~AS.

Data shown in Figs. :2 to 4 were plotted to demon­strate the correlation between the GUS activity in mature seeds and the ratio of OAS concentration to the

c Q)

(3 .... a. OJ E -­c

~ o E c

2.5~-------------------------------'

2

1.5

04--L--~------r-----~-----4

0.1 0.2 0.3 0.4 0.5

OAS/Met Fig. 5. Positive correlation between GUS activity driven by the f3 subunit gene promoter and [GAS] / [Met] ratio in the si­liques of transgenic A. thaliana. Data from Figs. :2 to 4 were used for the calculation of the correlation coefficient. The least square method was used for drawing the linear line best fit to the data.

Met concentration in 10 DAF siliques (Fig. 5), in which seed storage protein genes including the f3 subunit gene are actively transcribed (Heath et al. 1986; Hirai et al. 1995). There was a strong positive correlation between the GUS activity and endogenous [~AS] / [Met] ratio (r!. = 0.94). This strong correlation also indicates that Met and OAS act independently.

As previously reported, OAS is a key metabolite in the regulation of the expression of the f3 subunit gene under sulfur deficiency (Kim et al. 1999). Genes that are involved in sulfate assimilation including sulfate trans­porters, ATP sulfurylase, and adenosine 5' phosphosul­fate reductase genes, are known to be upregulated under sulfate deficiency and some of them respond to OAS applied exogenously (Smith et al. 1997; Hatzfeld and Saito 2000; Koprivova et al. 2000). These findings imply that OAS is a key metabolite responsible for the regula­tion of the sulfur assimilation pathway. There is little information available, on the other hand, on the role of Met in the regulation of gene expression. Met is the pre­cursor of S-adenosyl Met, which is then involved in the synthesis of polyamines and ethylene. Cysteine is a pre­cursor of glutathione, phytochelatins and a number of secondary metabolites. These compounds are important in a number of important processes including stress re­sponses. They are likely to play different roles in the sulfur assimilation pathway.

Although OAS and Met are several steps away from each other in the sulfur assimilation pathway in higher plants, key steps for the regulation of OAS and Met bio­synthesis are likely to be different. Expression in the chloroplasts of transgenic potato plants of a bacterial serine acetyltransferase, an enzyme responsible for the generation of OAS, has been reported to lead to in­creased levels of cysteine, suggesting that SAT is the limiting step of cysteine synthesis under normal condi­tions (Harms et al. 2000). Three isoforms of serine acet­yltransferases, each localized to the chloroplasts, cyto­sol, and mitochondria, have been described (Brunold and Suter 1982) and genes encoding three isoforms of serine acetyltransferase were cloned from A. thaliana by Noji et al. (1998), who demonstrated that these iso­forms, SAT-c, SAT-p, and SAT-m, are localized to the cytosol, chloroplasts, and mitochondria, respectively. Furthermore, it was found that SAT-c is sensitive to feedback inhibition by L-cysteine, unlike SAT-p and SAT-m (Noji et al. 1998). The increase of the content of OAS under sulfate deficiency may be explained by the release of serine acetyltransferase from feedback inhibi­tion by L-cysteine and this may occur in the cytosol.

It was demonstrated that posttranscriptional regula­tion of cystathionine )I-synthase encoded by the MT01 gene, plays a critical role in the regulation of methionine concentration in A. thaliana (Chiba et al. 1999). The

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Met and OAS Regulation of the ~ Subunit Gene 9:3

mtol-l mutation affects the content of cystathionine, but not that of homoserine (Inaba et al. 1994). Our observa­tion showing that the OAS content was not affected by the mtol-l mutation is in agreement with these findings.

We suggest that the regulation of the sulfur assimila­tion pathway may have two major blocks: one down to cysteine biosynthesis, and the other to Met synthesis. It appears that the pathway down to the synthesis of cys­teine is regulated by OAS and strongly influenced by sulfur nutritional conditions, whereas the latter pathway from cysteine to Met is not appreciably affected (Figs. 2 and 3). Thus, such diverse modes of regulation may be necessary for the sulfur assimilation pathway to fulfill its role.

It was reported that cysteine plays an important role in sulfur deficiency-regulated gene expression (Bolchi et al. 1999). Obviously. this does not seem to apply to the regulation of seed storage proteins because cysteine ap­plication to soybean cotyledon did not evidently affect seed storage protein composition (Kim et al. 1999). The involvement of glutathione in the regulation of the sulfur assimilation pathway has also been suggested (Saito 2000). Further studies on the metabolic regulation of the sulfur assimilation pathway will be necessary.

Acknowledgments. The authors thank Derek Goto (Hokkai­do University) for critical reading of the manuscript. This work was supported in part by grants from the Iijima Memorial Foun­dation for the Promotion of Food Science and Technology (Japan) to H.K.. from the Ministry of Education. Science, Sports and Culture of Japan for the Japanese Junior Scientists to M.Y.H. (No. 3639). for the Promotion of Science to T.F. (No. 09760053) and for Scientific Research on Priority Areas (B) to T.F. and S.N., Molecular Mechanisms of Storage Activity in Plants. M.Y.H. and H.K. were supported by the Japan Society of Promotion of Science for Young Scientists and Yoneyama Memorial Rotary Foundation. respectively.

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