Institute of Life Sciences, Ajinomoto Co., Inc., Kawasaki ... · 5 INTRODUCTION Photorespiration is a metabolic pathway in which glycolate-2-phosphate is produced by the oxygenase

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Daisuke Igarashi

Institute of Life Sciences, Ajinomoto Co., Inc., Kawasaki 210-8681, Japan

Tel. +81-44-244-5643

Fax +81-44-244-9617

E-mail [email protected]

Area Bioenergetics and Photosynthesis

Plant Physiology Preview. Published on September 1, 2006, as DOI:10.1104/pp.106.085514

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Glutamate:glyoxylate aminotransferase (GGAT) modulates amino acid contents

during photorespiration

Running head: Photorespiratory GGAT modulates amino acid contents

Daisuke Igarashi*, Hiroko Tsuchida, Mitsue Miyao, Chieko Ohsumi

Institute of Life Sciences, Ajinomoto Co., Inc., Kawasaki 210-8681, Japan (D.I., C.O.)

National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan (H.T., M.M.)



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Financial source

Part of this work was performed in the project “Development of Fundamental

Technologies for Controlling the Production of Industrial Materials by Plants,” which

was supported by the New Energy and Industrial Technology Development Organization

(NEDO), Japan.

*Corresponding author:

E-mail [email protected]

Tel. +81-44-244-5643

Fax +81-44-244-9617

Institute of Life Sciences, Ajinomoto Co., Inc., 1-1 Suzuki-cho Kawasaki-shi 210-8681,

Japan



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ABSTRACT

In photorespiration, peroxisomal glutamate:glyoxylate aminotransferase (GGAT)

catalyzes the reaction of glutamate and glyoxylate to produce 2-oxoglutarate and glycine.

Previous studies demonstrated that alanine aminotransferase-like protein functions as a

photorespiratory GGAT. Photorespiratory transamination to glyoxylate, which is

mediated by GGAT and serine glyoxylate aminotransferase (SGAT), is believed to play

an important role in the biosynthesis and metabolism of major amino acids. To better

understand its role in the regulation of amino acid levels, we produced 42 GGAT1

overexpression lines that express different levels of GGAT1 mRNA. The levels of free

serine, glycine, and citrulline increased markedly in GGAT1 overexpression lines

compared with levels in the wild-type, and levels of these amino acids were strongly

correlated with the levels of GGAT1 mRNA and with the GGAT activity in the leaves.

This accumulation began soon after exposure to light and was repressed under high levels

of CO2. Light and nutrient conditions both affected the amino acid profiles;

supplementation with NH4NO3 increased the levels of some amino acids compared with

the controls. The results suggest that the photorespiratory aminotransferase reactions

catalyzed by GGAT and SGAT are both important regulators of amino acid contents.



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INTRODUCTION

Photorespiration is a metabolic pathway in which glycolate-2-phosphate is produced by

the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco;

Leegood et al., 1995). At current atmospheric concentrations of O2 and CO2,

photorespiration in C3 plants dissipates more than 25% of the carbon fixed by means of

photosynthesis (Sharkey, 1988). Thus, photorespiration is often considered to be a

wasteful process. However, photorespiration may be a mechanism for mitigation of the

photoinhibition that can occur under high light levels, drought, and salt stress (Kozaki

and Takeba 1996; Hoshida et al., 2000; Wingler et al., 2000).

The photorespiratory nitrogen cycle contributes to the metabolism of certain amino

acids (Gln, Glu, Ser, and Gly). Nitrogen is incorporated into the photorespiratory C2

cycle through transamination of glyoxylate to glycine. Peroxisomal glyoxylate

aminotransferases play a central role within the photorespiratory pathway. The reactions

in this pathway are catalyzed by GGAT and SGAT. In Arabidopsis, it was reported that

alanine 2-oxoglutarate aminotransferase (AOAT)-like protein (Igarashi et al., 2003;

Liepman and Olsen, 2003) and alanine glyoxylate aminotransferase (AGT)-like protein

(Liepman and Olsen, 2001) function as photorespiratory GGAT and SGAT, respectively.

In the conversion of glycine to serine, ammonium and CO2 are released. Glutamine

synthetase 2 (GS-2) and ferredoxin-dependent (Fd-) GOGAT effectively refix the

released ammonium in plastids. It has been estimated that the production of ammonium

by photorespiration is an order of magnitude greater than the primary assimilation of

nitrogen resulting from nitrate reduction (Keys et al., 1978). That is, photorespiration

appears to be an essential process in the control of amino acid biosynthesis and

metabolism.

Serine and glycine are involved in protein biosynthesis and serve as precursors in a

variety of important biosynthetic pathways, including phospholipid synthesis (serine) and

purine formation (glycine). Serine and glycine are synthesized through two main

pathways. The first pathway involves transamination of glyoxylate into glycine, which is

in turn converted into serine. A source of glyoxylate is provided by oxidation of glycolate

during the photorespiratory cycle or the conversion of isocitrate to succinate during the

glyoxylate cycle involved in the degradation of fatty acids. The second pathway is



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connected with glycolysis and leads to serine formation from 3-phosphoglycerate.

Glycine is obtained from serine through a serine hydroxymethyltransferase (SHMT)

catalyzed reaction (Bourguignon et al., 1999). In photosynthetic tissues, photorespiration

is assumed to be a major pathway for serine and glycine synthesis because of the high

flux of carbon through this cycle (Gerbaud and Andre, 1979; Tolbert 1980; Somerville

and Somerville, 1983). In nonphotosynthetic tissues (e.g., seeds or roots), serine and

glycine are synthesized mainly though glycolysis and the glyoxylate cycle. However,

detailed regulation mechanisms of biosynthesis of these amino acids are unclear.

In the present study, we investigated photorespiratory transamination using GGAT1

overexpression and knockout lines, and estimated the function of GGAT in the regulation

of amino acid metabolism linked to photorespiration.



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RESULTS

Expression analysis

Genomic sequence information indicated that there are four alanine aminotransferase-

like genes in Arabidopsis (Igarashi et al., 2003). Two of them (GGAT1; At1g23310 and

GGAT2; At1g70580) that encode proteins that contain peroxisome targeting signal 1

(PTS1) were identified as encoding photorespiratory GGAT proteins (Igarashi et al.,

2003; Liepman and Olsen, 2003). To better characterize the function of these genes, we

examined the tissue specific expression patterns of two GGAT genes (GGAT1 and

GGAT2) using promoter-GUS system. We also examined the diurnal changes in mRNA

levels.

The GUS stain was strongly detected in leaves both of GGAT1- and GGAT2–GUS

seedlings (Fig. 1A), which suggests that GGAT1 and GGAT2 function as

photorespiratory GGAT enzymes. In GGAT1–GUS seedlings, GUS signals were also

strongly detected in roots (Fig. 1A), suggesting that the role of GGAT1 is not restricted to

photorespiration. The function of root peroxisomes has not been identified, but in

nonphotorespiratory tissue (roots) and in darkness, GGAT1 may catalyze the

aminotransferase reaction. At night, serine contents increased in a GGAT1-knockout line

(Igarashi et al., 2003), supporting this hypothesis.

After light exposure, the levels of GGAT1 mRNA decreased and those of GGAT2

mRNA increased rapidly (Fig. 1B). We previously reported that GGAT1 knockout line

(ggat1-1) cannot grow normally in air and recovered under high CO2 conditions, and

concluded that GGAT1 is a major (Igarashi et al., 2003). However the enzymatic activity

of GGAT remained constant during the day (Fig. 2A), suggesting that GGAT1 and

GGAT2 may function redundantly in leaf photorespiration.

Generation of GGAT1 overexpression lines

Detailed analysis of GGAT1 overexpression and knockout plants is necessary for

characterization of the regulatory system for amino acid metabolism that depends on

photorespiration. We previously isolated a GGAT1 knockout line (ggat1-1) from tag-

insertion lines (Igarashi et al., 2003). In the present study, we generated 42 GGAT1

overexpression lines by introducing the GGAT1 genomic region. To characterize the



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relationship between GGAT1 activity and amino acid metabolism, it is essential to isolate

many GGAT1 overexpression lines that have different GGAT activities. We selected the

native GGAT1 promoter rather than a constitutive promoter to control GGAT1

expression. Furthermore, by transferring the 5'-region of GGAT1 to the left border side

of T-DNA, we successfully isolated many transgenic lines that expressed various levels

of GGAT1 mRNA. Levels of GGAT1 mRNA and GGAT activities in the GTox-17

GGAT1 overexpression line were higher than those in the wild-type (Fig. 2A,B). The

diurnal changes in mRNA levels (Fig. 1) remained apparent (Fig. 2B). Diurnal GGAT

activities remained stable in both the wild-type and the GTox-17 line (Fig. 2A).

Correlation of amino acid contents, GGAT activities, and GGAT1 mRNA levels

We selected nine transgenic lines that expressed a wide range of GGAT1 mRNA levels

and determined their amino acid contents, GGAT activities, and GGAT1 mRNA levels.

As shown in Figure 3, there was a strong correlation between GGAT activity and the

level of GGAT1 mRNA (R=0.84). In addition, the serine (R=0.97), glycine (R=0.74), and

citrulline (R=0.94) contents were also strongly correlated with GGAT activity. Table I

presents the correlation values between GGAT1 activity and amino acid contents. Other

than serine, glycine, and citrulline, no significant correlations were observed. The results

suggest that GGAT activity is the limiting factor for synthesis of serine, glycine and

citrulline

Amino acid accumulation

All 42 GGAT1 overexpression lines accumulated serine, glycine, and citrulline (Fig. 4).

Most of the increase in total amino acid level compared with the wild-type was accounted

for by increased serine in the GGAT1 overexpression lines and by increased glutamate in

the knockout line (ggat1-1) because the ratios of serine and glutamate to total amino acid

in the wild-type were 0.1 and 0.2, respectively. Following two results strongly suggest

that the alteration of amino acid contents resulted directly from overexpression of the

GGAT1 genes. First, there were strong correlations between GGAT activity and the levels

of GGAT1 mRNA, serine, glycine, and citrulline (Table I). Second, all transgenic lines

accumulated serine, glycine, and citrulline (Fig. 4). Furthermore, the accumulation of



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serine suggested that active conversion of glycine to serine is occurring and that SGAT

activity is limited. The increased total amino acid contents in the overexpression lines

reveal the possibility of improving nitrogen use efficiency by creating GGAT1

overexpression lines.

Serine accumulation in the GGAT1 overexpression line of rice

We generated transgenic rice that expressed the Arabidopsis GGAT1 cDNA. The

expression of the GGAT1 cDNA under the control of the Arabidopsis GGAT1 promoter

did not lead to overproduction of GGAT1 in transgenic rice (data not shown). Instead,

the use of the rice Cab promoter, which is known to direct high levels of expression in

photosynthetic tissues (Sakamoto et al., 1991) successfully increased GGAT1 mRNA

levels and serine contents in the leaves, as was the case in Arabidopsis (Fig. 5).This

suggests that basic mechanisms for amino acid metabolic regulation by means of

photorespiration processes may be similar between dicots and monocots.

Photorespiration and amino acid accumulation

To confirm that the increased levels of serine, glycine, and citrulline in GGAT1

overexpression lines resulted directly from alterations in the photorespiration pathways,

we analyzed the amino acid contents of transgenic plants grown under conditions that

would suppress photorespiration (3,000 ppm). GGAT1 overexpression line GTox-17

accumulated serine, glycine, and citrulline at 13.4, 3.2, and 2.4 times the respective levels

observed in the wild-type (Col-0) under normal CO2 conditions, versus 5.2, 1.9, and 1.9

times the respective levels in the wild-type under high CO2 conditions (Fig. 6). The

reduction of accumulation levels under high CO2 conditions in GGAT1 overexpression

lines also observed, indicating that accumulation of serine, glycine and citrulline

depended on photorespiration. Furthermore serine was not accumulated in GGAT1

knockout line, suggesting that almost of serine is synthesized through photorespiration.

Diurnal changes in amino acid levels

To investigate the relation of amino acid metabolisms with photorespiration, we

analyzed diurnal changes in amino acid.



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The levels of serine, glycine, and citrulline increased during the morning, remained

relatively constant during the afternoon, and then declined after sunset in the wild-type

(Fig. 7A). In contrast, the levels of these amino acids continued to increased throughout

the daytime in the GGAT1 overexpression lines. Serine increased most dramatically, to

20 times the level in the wild-type. The increase in serine depended on GGAT activity in

the GGAT1 overexpression line during the day (Fig. 3, 4). Therefore, it is possible that

GGAT activity is a limiting factor for serine biosynthesis in the wild-type.

Since GGAT activity did not change during the daytime (Fig. 2A), it is concluded that

the substrate supply is the limiting factor for serine biosynthesis in the GGAT1

overexpression lines. This hypothesis is supported by the observation that overexpression

line GTox-4, which expressed the highest level of GGAT1 mRNA, and two other lines

(GTox-17 and GTox-24) with levels nearly as high, showed a similar rate of increase in

serine (Fig. 7B).

Glutamate and alanine decreased in the GGAT1 overexpression lines and increased in

the knockout line (ggat1-1), suggesting that these amino acids as amine donors for the

GGAT reaction. Glutamine and aspartate accumulation in the knockout line would result

from excess amino groups that were transferred into pools of these amino acids.

Differences in nitrogen source affect the amino acid profiles

Amino acid profiles in higher plants are modulated by the composition of the nitrogen

source exogenously supplied to the growth medium (Raab and Terry, 1995). We

therefore compared the amino acid contents between the wild-type and the GGAT1

overexpression lines grown in different nutrient solutions.

As shown in Table II, the GGAT1 overexpression lines grown in PNS medium or in

PNS + NH4NO3 medium both accumulated serine, glycine, and citrulline compared with

the wild-type. Glutamine and asparagine accumulated in plants grown in the PNS +

NH4NO3 medium, but not in plants grown in the PNS medium. Although arginine was

not detected in plants grown in PNS medium, it accumulated in the GGAT1

overexpression lines grown in PNS + NH4NO3 medium. Other than these amino acids,

the different nitrogen sources did not appear to affect the amino acid contents. These

modulations in the amino acid profiles appear to reflect the efficiency of ammonium use.



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Amino acid accumulation in sink tissues

We investigated the effects of GGAT1 overexpression on amino acid profiles in the sink

tissues. In the GGAT1 overexpression lines, serine and glycine both accumulated much

more in sink tissues than was the case in the wild-type and knockout lines (ggat1-1; Fig.

8). However, serine and glycine levels in the ggat1-1 appeared to be comparable to those

in the wild-type in both stmes and silique. Although the mechanism is unclear, it appears

that alteration of metabolic regulation by photorespratory GGAT improved nutrient levels

in the sink tissues.



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DISCUSSION

Accumulation of serine, glycine, and citrulline in GGAT1 overexpression lines

More than 25% of the carbon fixed during photosynthesis may be used up by

photorespiration (Sharkey, 1988). In addition, leaf mitochondria generate ammonium at

an extraordinary rate during photorespiration, as much as 50 times the rate of nitrate

reduction (Ogren, 1984). This toxic ammonium is efficiently reassimilated into glutamine

and glutamate by GS-2 and Fd-GOGAT, and these are then used to produce nitrogenous

compounds needed for plant growth. Thus, photorespiratory amino acid metabolic

pathways appear to be important for nitrogen metabolism in plants.

GGAT1 overexpression lines accumulated high levels of serine, glycine, and citrulline

compared with the wild-type. High positive correlations between levels of these amino

acids, GGAT1 expression, and GGAT enzymatic activities were observed under

photosynthetic conditions (Fig. 3). These amino acids accumulated after transfer of the

plants into the light (Fig. 7). In contrast, the rate of accumulation decreased under high

CO2 (Fig. 6). These results demonstrate that GGAT1 overexpression lines accumulate

these amino acids at rates determined by the rate of photorespiration. Although the level

of GGAT1 mRNA declined from morning to midday in both the wild-type and the GTox-

17 GGAT1 overexpression line (Fig. 2B), the decrease was much larger in GTox-17 and

GGAT activity did not change in either group of plants. This might result from GGAT2

accumulation after light irradiation and the stability of GGAT proteins. Throughout the

day, serine, glycine, and citrulline increased in the GTox-17 GGAT1 overexpression line

(Fig. 7A) even though GGAT activity did not increase (Fig. 2A), suggesting that shortly

after light exposure, the limiting factor for accumulation of these amino acids is not

GGAT activity but rather the supply of substrate for the GGAT reactions during

photorespiration. Thus, when the rate of photorespiration is high, GGAT activity may be

a limiting factor in the photorespiratory cycle.

SGAT also plays a major role in controlling the flux of carbon toward glycine, and in

supplying the substrate for serine synthesis (Somerville and Ogren, 1980). In this way

GGAT and SGAT could function cooperatively in the production of the photorespiratory

amino acid glycine.



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Conversion of glycine into serine and energy consumption

The positive correlation between serine content and GGAT activity (Fig. 3) was

stronger than that for glycine, which is synthesized directly from the GGAT reaction. The

absolute serine content was also much higher than that of glycine (Table II). The

conversion of glycine into serine is catalyzed by glycine decarboxylase (GDC) and

SHMT and is accompanied by the generation of NH3, CO2, and NADH (Oliver and

Raman, 1995). GDC is extremely abundant in mitochondria isolated from leaves (Douce

and Neuburger, 1999). Therefore, the conversion of glycine into serine should be a stable

and active reaction. NADH generated by this reaction might be translocated to the

peroxisomes, where it would serve as a substrate for the reduction of hydroxypyruvate to

glycerate by hydroxypyruvate reductase (Raghavendra et al., 1998). In addition, recent

research has suggested that ammonium and NADH generated during glycine conversion

for glutamine and citrulline synthesis is catalyzed by GS-2, carbamoylphosphate

synthetase, and ornithine carbamoyltransferase in the mitochondria (Taira et al., 2004;

Linka and Weber, 2005). Glycine-driven oxidative phosphorylation should yield roughly

three ATP per glycine consumed. In the conversion of ornithine to citrulline, one ATP is

consumed for glutamine synthesis and two ATP are consumed for carbamoylphosphate

synthesis, allowing tight coupling of the phosphorylation and synthesis reactions (Taira et

al., 2004). Citrulline is converted into arginine (Ludwig, 1993). Because the citrulline

contents increased greatly after transfer of plants into the light in GGAT1 overexpression

lines and in the wild-type (Fig. 7A), the arginine content in GGAT1 overexpression line

grown in a medium with supplemental ammonium was higher than that in the wild-type

(Table II). Our results are thus consistent with reported pathways for the metabolism of

nitrogen released by photorespiration. The absolute contents of citrulline and arginine

that accumulated in the GGAT1 overexpression line were low compared with those of

glutamine and glutamate, demonstrating that mitochondrial reassimilation of ammonium

is a minor pathway.

Analysis of the function of GGAT in monocot

Transgenic rice that expressed Arabidopsis GGAT1 also accumulated serine. Rice also

has predicted GGAT-like genes in both the O. japonica (BAC20747) and O. indica



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(AAO84040) cultivars (International Rice Genome Sequencing Project, 2005). The

amino-acid sequence of the protein encoded by Arabidopsis GGAT1 is 80% similar to

that of the rice GGAT1-like proteins. These two groups of proteins contain a

serine/arginine/methionine group at the C-terminus that is a conserved PTS1. These

sequence characteristics support the hypothesis that the rice GGAT1-like proteins

function as photorespiratory GGATs. This suggests that the regulation of

photorespiratory glyoxylate aminotransferase is similar in a dicot (Arabidopsis) and a

monocot (rice). However, the degree of serine accumulation in rice was not remarkable

compared with that in Arabidopsis, and other than serine, we detected no marked

differences in amino acid accumulation between the wild-type and the GGAT1

overexpression line of rice. This could result from instability in GGAT1 originating in a

heterologous expression system, slight differences in the metabolite flux during

photorespiration, and small absolute levels of GGAT1 mRNA.

Photorespration and nitrogen

Nitrogen, an essential nutrient for plant growth, is primarily taken up by roots in the

form of nitrate and is reduced to ammonium by nitrate reductase and nitrite reductase. In

most higher plants, ammonium is assimilated into glutamine through the cooperative

activity of GOGAT and GS. These reactions represent the major pathway for the

assimilation of ammonium into amino acids. At elevated CO2 concentrations,

photorespiration is limited and the assimilation of nitrogen is inhibited in the shoots of

dicotyledons (e.g., Arabidopsis) and monocotyledons (e.g., wheat; Bloom et al., 2002;

Rachmilevitch et al., 2004). Thus, under normal conditions, photorespiration is assumed

to contribute to effective use of nitrogen.

Our results demonstrated that in an ammonium-rich medium, levels of Glutamine,

asparagine, and arginine increased in the both of the wild-type and the GGAT1

overexpression lines (Table II), suggesting that these amino acids form a nitrogen pool

under nitrogen-rich conditions. However, the increase in levels of these amino acids in

the GGAT1 overexpression line in response to supplemental ammonium was much higher

than the increase in the wild-type. This suggests that increased release of ammonia during

the conversion of glycine into serine and uptake of ammonium by the roots, ammonium



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increases to excessive levels and would be assimilated into less-toxic glutamine,

asparagine, and arginine. The high accumulation of total amino acids in the GGAT1

overexpression line may show that improvement in GGAT activity in this line can

enhance photorespiration and the efficiency of nitrate and ammonium assimilation.

Substrates for GGAT reaction

The analysis of GGAT- and SGAT-deficient mutants and of recombinant proteins

indicated that alanine is a substrate for both GGAT and SGAT (Murray et al., 1987;

Liepman and Olsen, 2001, 2003; Igarashi et al., 2003). GGAT catalyzes an AGT reaction

(Noguchi and Hayashi, 1981; Nakamura and Tolbert, 1983; Yu et al., 1984, Igarashi et

al., 2003; Liepman and Olsen, 2003). However, a high Km value of SGAT for alanine

(101.2 mM) indicates that alanine may not be a physiological substrate for SGAT

(Liepman and Olsen, 2001).

The diurnal glutamate and alanine profiles differed between the knockout line (ggat1-1)

and the overexpression line (Fig. 7A). From morning (8:00) to night (0:00), these amino

acids increased in ggat1-1, and decreased in the overexpression line. Both glutamate and

alanine are used as amine donors for transamination to glyoxylate in the GGAT reaction.

Estimates based on whole-leaf labeling analysis suggested that alanine contributed three

times more amino groups to photorespiratory glycine formation than glutamate did

(Betsche, 1983), whereas another study based on feeding metabolites to purified

peroxisomes concluded that glutamate contributed more to glyoxylate transamination

than alanine did (Yu et al., 1984). The cooperative change of glutamate and alanine in the

ggat1-1 knockout line and the GGAT1 overexpression line suggests that both amino acids

contribute to transamination for glycine formation.

Amino acid accumulation in sink tissues

We showed that serine and glycine contents increased in sink tissues (Fig. 8). Although

this may be a result of photorespiration in stem and silique, accumuration level of serine

was higher than that of in leaves, suggesting that amino acids in the source tissues would

be directly transported to sink tissues and concentrated. Because photorespiration is

involved in photosynthesis and metabolisms of nitrogen and amino acid, and protection



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against stress, it is a proposed target for genetic engineering to improve crop productivity.

However, further analysis of the flux of photorespiratory metabolites and of the transport

and accumulation mechanisms between source and sink tissues will be necessary to

permit effective improvement of crop production by engineering of the genes involved in

photorespiration.

In summary, our analysis of the ggat1-1 knockout and GGAT1 overexpression lines

suggested that GGAT activity is directly involved in the regulation of glycine and serine

levels. Furthermore, the total amino acid content and levels of other major amino acids

differed markedly between these two lines, suggesting that GGAT may regulate the

biosynthesis and metabolism of fundamental amino acids.



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MATERIALS AND METHODS

Plant materials and growth conditions

Arabidopsis thaliana L. (Col-0) cultures were grown at photosynthetically active

radiation (PAR) of 70 µmol m-2 s-1 on a 16:8-hr L:D photoperiod at 22 °C unless

otherwise indicated. A growth camber (EYELA FLI-301NH-CL) was used for the

analysis of plants grown under high CO2 concentrations. Plants were grown on sterile

plant nutrients and sugar (PNS) medium (Haughn and Somerville, 1986) in petri dishes

containing 0.8% (w/v) agar.

To compare the amino acid contents between the wild-type and the GGAT1

overexpression lines grown under different nitrogen sources, these plants were grown on

PNS medium with or without supplementation with 10 mM NH4NO3. PNS medium

contains 5 mM KNO3 and 2 mM Ca(NO3)2 as nitrogen sources and an additional 10 mM

NH4NO3 almost doubled nitrogen in the medium. The plants were grown for 2 weeks on

PNS medium or on PNS + NH4NO3 medium, and the aboveground tissues were collected

for amino acid analysis.

To determine the amino acid contents in the plants grown under high CO2 conditions or

in the siliques and stems, the plants were grown in soil watered with PN medium at PAR

of 120µmol m-2 s-1. Arabidopsis siliques (9 to 11 days after flowering), stems (cut 15 cm

above the roots),and fully expanded leaves were harvested for analysis.

Rice (Oryza sativa L. cv ‘Kitaake’) was planted in soil watered with 1,000-fold diluted

Hyponex (Hyponex, Osaka) and grown under natural light conditions on a 14:10-hr L:D

photoperiod at 23°C day/18 °C night cycle in a greenhouse (Tsuchida et al., 2001).

Expression analysis of GGAT

For reverse-transcriptase PCR (RT-PCR), total RNA was isolated from leaves using an

RNeasy Plant Mini Kit (Qiagen). First-strand cDNA synthesis was conducted with an

oligo dT 12-18 primer (GE Healthcare) and reverse transcriptase, superscript II

(Invitrogen). Real-time PCR reactions were performed using an Applied Biosystems

Prism 7700 Sequence Detector. Specific primers for GGAT1 (5'-

TTCTTCTTCTGAACGACTATTGTG-3' and 5'-



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GAATAGGGCAAAGAGAAAGAGTG-3') and for GGAT2 (5'-

TCACTTCTTCTTCTTTCACGACA-3' and 5'-GAGTTTGACACAGAGTAGGACCA-

3') were used.

For rice analysis, specific primers for GGAT1 (5'-TGAAAGCAAGGGGATTCTTG-3'

and 5'-GACGTTTTTGCAGCTGTTGA-3') were used for quantification of the

introduced gene.

Real-time PCR reactions were performed in duplicate using 0.9 µM of each primer and

1 x SYBR green PCR master mix (Applied Biosystems) in a 50-µL volume. Relative

differences were determined using the deltaCt method described by the manufacturer.

Construction of plasmids and transformation of Arabidopsis

The promoter regions were isolated by means of PCR using specific primers for GGAT1

(5'-CAATAACAATGCAAAGTTAAGATTCGGATC-3' and 5'-

GACGGATCCCATTTATGTTCACTCTGAACC-3') and for GGAT2 (5'-

GACAAGCTTTGAGTTCGGACACGGCTCTA-3' and 5'-

GACGGATCCCATTTCCAGGATTTACTTTTC-3'). The amplified fragments were

digested by BamHI (GGAT1) or HindIII/BamHI (GGAT2) and the fragments (1843 bp for

GGAT1 and 2k bp for GGAT2) were cloned into the 5'-end of the GUS gene in vector

pBI101.

For generation of a GGAT1 overexpression line of Arabidopsis, a 5089-bp genomic

fragment of GGAT1, that extends 5'-upstream (1843 bp) from the translational initiation

and 3'-downstream (612 bp) from the point of translational termination, was isolated by

means of PCR using specific primers (5'-

CAATAACAATGCAAAGTTAAGATTCGGATC-3' and 5'-

GCTTCTTCTCAACCATCGTCACC-3'). The amplified fragment was cloned into vector

pBI121 between the HindIII and EcoRI sites and introduced into the wild-type (Col-0) ,

or an GGAT1 knockout line (ggat1-1) by means of Agrobacterium tumefaciens–mediated

transformation. Two of 42 lines are ggat1-1 background and others are wild-type

background.

Generation of a GGAT1 overexpression line of rice



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Arabidopsis GGAT1 cDNA was isolated by means of RT-PCR using specific primers

(5'-GGCTCTAGATGGCTCTCAAGGCATT-3' and 5'-

GCCGAGCTCTCACATTTTCGAATAA-3'). The amplified fragment was fused to the

Cab promoter (Sakamoto et al., 1991) and cloned into the vector pIG121Hm. The

resultant plasmid was introduced into calli derived from rice by means of A. tumefaciens–

mediated transformation.

Histochemical staining for GUS activity

Histochemical staining for GUS activity was carried out as described by Jefferson et al.

(1987), but with the following modifications. After transgenic plants had been grown for

2 weeks on the PNS–agar plates, plants were harvested and incubated overnight in GUS

staining solution [100 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.1% (v/v) triton

X-100, 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide and 1 mM X-Gluc]

at 37 °C. After staining, samples were treated with 70% ethanol to remove chlorophyll

from the tissues.

Enzyme assays

Aminotransferase activity was assayed as described by Hørder and Rej (1983), but with

the following modifications. The aboveground tissues of seedlings grown on PNS–agar

plates at PAR of 70 µmol m-2 s-1 were harvested, frozen, and ground into a fine powder

using an MM 300 mixer (Qiagen). The ground powder was added to 0.5 mL of grinding

buffer [100 mM Tris-HCl (pH 7.3) and 10 mM dithiothreitol] and gently mixed to ensure

complete suspension. The suspension was then centrifuged (13,000 x g for 10 min at 4

°C) to remove insoluble material. The resulting supernatant (0.5 mL) was applied to a

UFV5BGC00 Ultrafree filter (Millipore) for concentration and desalination. The total

protein concentration was assayed using a Bio-Rad protein assay system

GGAT activity was measured by coupling the reduction of 2-oxoglutarate by NADH in

a reaction catalyzed by GDH. The reaction was assayed in a mixture containing 100 mM

Tris-HCl (pH 7.3), 20 mM glutamate, 1 mM glyoxylate, 0.18 mM NADH, 0.11 mM

pyridoxyal 5-phosphate, 83 mM NH4Cl, and 0.3 U GDH in a final volume of 0.6 mL.



20

Activity was assayed spectrophotometrically by monitoring NADH oxidation at 340 nm.

The GGAT assays used 50 µg of crude extract. All reactions were performed at 30 °C.

Measurement of amino acid contents

Amino acids were extracted from the aboveground tissues of the plants in 80% ethanol

at 80 °C. After evaporation, dried samples were dissolved in 0.02 N HCl. Amino acid

contents were determined using an L-8800 amino acid analyzer (Hitachi). Briefly, amino

acids, separated by cation-exchange chromatography, were detected

spectrophotometrically after postcolumn reaction with ninhydrin reagent (Noguchi et al,

2006).

ACKNOWLEDGMENTS

We thank Kiyoshi Miwa, Yutaka Ishiwatari, Hiroyuki Kiyohara, and Ai Koga for

helpful discussion and technical assistances during this work.

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24

FIGURE LEGENDS

Figure 1

Expression of GGAT1 and GGAT2 in Arabidopsis.

A, Analysis of tissue-specific expression by means of histochemical staining of 2-week-

old seedlings harboring promoter–GUS constructs.

B, Diurnal changes in mRNA contents

Time-points were 2, 8, and 15 hours after exposure to light and 2 and 7 hours after

darkness resumed. Relative amounts of mRNA were measured by means of RT-PCR and

calculated using the deltaCt method. Seedlings grown on two independent plates were

used for samples. Values represent means (black circles) and maximum and minimum

values (white circles). Within each graph, values are normalized by expressing them as a

multiple of the lowest value recorded during the day.

Figure 2

Diurnal changes in GGAT enzymatic activities and mRNA levels in the GGAT1

overexpression line of Arabidopsis.

Seedlings of Col-0 and the GTox-17 GGAT1 overexpression line were grown on PNS–

agar plates for 2 weeks. Aboveground tissues were harvested at each sampling time.

A, Durnal change in GGAT enzymatic activities. The methods of enzyme assay is

described in the text.

B, Relative amounts of mRNA were measured by means of real-time PCR and calculated

using the deltaCt method same as described in legend of figure 1.

Figure 3

Correlations between amino acid contents, GGAT activities, and GGAT1 mRNA levels.

Nine independent GGAT1 overexpression line of Arabidopsis that exhibited a wide range

of levels of GGAT1 mRNA were grown on PNS–agar plates for 2 weeks. Aboveground

tissues were harvested at midday and amino acid contents, GGAT activities, and GGAT1

mRNA levels were determined. The assay methods are described in the text. Data are

only presented for correlations that were significant in Table I. Values represent the



25

relative amounts compared with the value for the wild-type (Col-0). R means correlation

coefficient.

Figure 4

Amino acid contents in the GGAT1 overexpression line of Arabidopsis.

Forty-two independent GGAT1 overexpression lines that expressed various levels of

GGAT1 mRNA, as well as a knockout line (ggat1-1) and the wild-type (Col-0), were

grown on PNS–agar plates for 2 weeks. Aboveground tissues were harvested at midday

and amino acid contents were determined. In the graph of total amino acid contents, dark

and white bars indicate serine and other protein composition amino acid with citrulline,

respectively.

Figure 5

Levels of GGAT1 mRNA and serine accumulation in a GGAT1 overexpression line of

rice.

Ten independent T2 transgenic plants derived from four independent GGAT1

overexpression lines (lines 27-6, 31-4, 35-3, and 39-3) and the wild-type were grown on

soil for 6 weeks. The middle sections of the leaves were harvested at midday and GGAT1

mRNA levels and serine contents were determined. Relative amounts of mRNA were

measured by means of real-time PCR and calculated using the deltaCt method. Within

each graph, values are normalized by expressing them as a multiple of the lowest value

(line 27-6) recorded in GGAT1 overexpression line of rice. The n.d. means not detected.

Figure 6

Effects of CO2 concentration on the accumulation of amino acids.

Arabidopsis plants of the wild-type (Col-0), GGAT1 knockout line (ggat1-1), and GGAT1

overexpression line (GTox-17) were grown in soil under normal (3,000 ppm) or high

(300 ppm) CO2 levels for 3 weeks. Aboveground tissues were harvested and amino acid

contents were determined.

Figure 7



26

Diurnal changes in amino acid contents.

Arabidopsis seedlings of the wild-type (Col-0), GGAT1 knockout line (ggat1-1), and

GGAT1 overexpression line (GTox-17) were grown on PNS–agar plates for 2 weeks.

Values represent the means from two dependent samples from dishes (line) and the

maximum and minimum values (each symbol).

A, Aboveground tissues were harvested at each time and amino acid contents were

determined. B, Serine levels in the wild-type, in the knockout line (ggat1-1), and in three

independent overexpression lines (GTox-4, -17 and -24).

Figure 8

Serine and glycine accumulation in stem and silique.

Arabidopsis plants of the wild-type (Col-0), GGAT1 knockout line (ggat1-1), and GGAT1

overexpression line (GTox-17) were grown in soil for 6 weeks. Stems were harvested 15

cm from the base. Siliques were harvested 9 to 11 days after flowering. All samples

including leaves were harvested at midday.

Table I

Correlations between GGAT activity and each amino acid that was assayed, as well as

between GGAT activity and the levels of GGAT1 mRNA.

Col-0 (wild-type) and GGAT1 overexpression lines (GTox) were grown and amino acid

contents and mRNA levels were determined using the methods described in the text.

Table II

Amino acids pool size in GGAT1 overexpression lines

Each line was grown on PNS medium or on PNS + 10mM NH4NO3 medium on agar

plates for 2 weeks. Aboveground tissues were harvested at midday and amino acid

contents were determined. Seedling were grown in 4 separate plates. Value represent the

average for each experiment, with the S.D. in parentheses.



27

Table I

amino acid correlation

Ser 0.97

Cit 0.94

Gly 0.74

Asn 0.49

Gln 0.46

Ala -0.21

Glu -0.40

Asp -0.49

mRNA 0.84

TableII

PNS nmol/mg FW Fold

Col-0 GTox-17 GTox-24 GGT-17 GGT-24

Asp 1.04 ( 0.04 ) 0.74 ( 0.04 ) 0.56 ( 0.10 ) Asp 0.72 0.54

Asn 0.28 ( 0.03 ) 0.33 ( 0.02 ) 0.35 ( 0.02 ) Asn 1.21 1.25

Glu 2.91 ( 0.10 ) 2.45 ( 0.05 ) 2.56 ( 0.10 ) Glu 0.84 0.88

Gln 1.01 ( 0.10 ) 0.89 ( 0.03 ) 0.96 ( 0.03 ) Gln 0.88 0.94

Ser 0.96 ( 0.03 ) 7.47 ( 0.15 ) 8.96 ( 0.24 ) Ser 7.76 9.31

Gly 0.11 ( 0.01 ) 0.57 ( 0.02 ) 0.68 ( 0.05 ) Gly 5.09 6.09

Ala 0.52 ( 0.10 ) 0.23 ( 0.05 ) 0.29 ( 0.01 ) Ala 0.45 0.56

Arg ND ND ND Arg - -

Cit ND 0.11 ( 0.01 ) 0.12 ( 0.00 ) Cit - -

PNS+10mM NH4NO3 nmol/mg FW Fold

Col-0 GTox-17 GTox-24 GGT-17 GGT-24

Asp 0.78 ( 0.10 ) 0.75 ( 0.10 ) 0.55 ( 0.10 ) Asp 0.96 0.71

Asn 0.77 ( 0.12 ) 2.16 ( 0.26 ) 2.56 ( 0.11 ) Asn 2.80 3.33

Glu 2.28 ( 0.15 ) 2.20 ( 0.14 ) 2.56 ( 0.23 ) Glu 0.97 1.12

Gln 2.20 ( 0.34 ) 4.55 ( 0.66 ) 5.43 ( 0.98 ) Gln 2.07 2.47

Ser 1.43 ( 0.13 ) 12.34 ( 0.34 ) 14.98 ( 1.26 ) Ser 8.61 10.45

Gly 0.60 ( 0.09 ) 3.22 ( 0.43 ) 3.30 ( 0.45 ) Gly 5.40 5.53

Ala 0.57 ( 0.05 ) 0.44 ( 0.11 ) 0.38 ( 0.02 ) Ala 0.77 0.67

Arg 0.55 ( 0.13 ) 1.13 ( 0.02 ) 1.25 ( 0.09 ) Arg 2.04 2.27

Cit 0.07 ( 0.01 ) 0.34 ( 0.02 ) 0.43 ( 0.04 ) Cit 5.03 6.30



















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