lsolation and Characterization of Clutamine …et al., 1993). ln higher plants, the genes encoding individual GS isoforms are regulated differently by environmental signals such as

Plant Physiol. (1996) 112: 987-996

lsolation and Characterization of Clutamine Synthetase Genes in CMamydomonas reinhardtii’

Qiang Chen’ and Carolyn D. Silflow*

Department of Genetics and Cell Biology (Q.C., C.D.S.), Plant Molecular Genetics lnstitute (Q.C., C.D.S.), and Department of Plant Biology (C.D.S.), University of Minnesota, St. Paul, Minnesota 551 O8

To elucidate the role of glutamine synthetase (CS) in nitrogen assimilation in the green alga Chbmydomonas reinhardtii we used maize CS1 (the cytosolic form) and CS2 (the chloroplastic form) cDNAs as hybridization probes to isolate C. reinhardfii cDNA clones. The amino acid sequences derived from the C. reinhardtii clones have extensive homology with CS enzymes from higher plants. A putative amino-terminal transit peptide encoded by the GS2 cDNA suggests that the protein localizes to the chloroplast. Cenomic DNA blot analysis indicated that CS1 is encoded by a single gene, whereas two genomic fragments hybridized to the CS2 cDNA probe. AI1 CS2 cDNA clones corresponded to only one of the two CS2 genomic sequences. We provide evidence that ammonium, nitrate, and light regulate CS transcript accumulation in green algae. Our results indicate that the level of CS1 transcripts is repressed by ammonium but induced by nitrate. The level of CS2 transcripts is not affected by ammonium or nitrate. Expression of both CS1 and CS2 genes is regulated by light, but perhaps through different mechanisms. Unlike in higher plants, no decreased level of CS2 transcripts was detected when cells were grown under conditions that repress photorespiration. Analysis of CS transcript levels in mutants with defects in the nitrate assimilation pathway show that nitrate assimilation and ammonium assimilation are regulated independentl y.

GS (EC 6.3.1.2) is a primary biological catalyst for the first step at which nitrogen is brought into cellular metabolism. It is a key enzyme of nitrogen metabolism found in a11 life-forms and functions in two essential biochemical reactions: ammonium assimilation and Gln biosynthesis. GS catalyzes the formation of Gln from ammonium and glutamate in the presence of ATP and Mg. Gln is then used by glutamate synthase or GOGAT (Fd-GOGAT, EC 1.4.7.1, or NAD(P)H-GOGAT, EC 1.4.1.14) to produce glutamate. The glutamate produced by the GS-GOGAT cycle is the nitrogen donor in the biosynthesis of essentially a11 cellular nitrogenous compounds.

In higher plants, GS enzymes can be classified into groups according to their localization within the cell, in-

Q.C. was supported by fellowships from the Plant Molecular Genetics Institute, University of Minnesota, and from the National Research Service (GM 17089). This work was partially supported by National Institutes of Health grant GM51995 to C.D.S.

Present address: Croptech Corp., 1861 Pratt Drive, Blacksburg, VA 24600.

* Corresponding author; e-mail [email protected]; fax 1- 612- 625-5754.

cluding the cytosolic form, GS1, and the chloroplastic form, GS2 (reviewed by Last, 1993; Lam et al., 1995). In addition, some legumes have another cytosolic GS form, GSn, which is present only in the root nodules (Last, 1993). Multiple GSl isoforms have been shown to be encoded by small gene families. However, in a11 higher plant species studied to date, only one isoform of GS2, which is encoded by a single nuclear gene, has been identified (Cullimore et al., 1984; Tingey et al., 1987; Peterman and Goodman, 1991; Li et al., 1993). ln higher plants, the genes encoding individual GS isoforms are regulated differently by environmental signals such as light, nitrate, and ammonium, and by the metabolic status of the cell (Hirel et al., 1987; Tingey et al., 1987,1988; Edwards and Coruzzi, 1989; Kozaki et al., 1991; Sukanya et al., 1994). The expression patterns of genes for different GS isoforms also show different tissue or cell specificity during plant development (Cullimore et al., 1984; Edwards et al., 1990; Brears et al., 1991; Kozaki et al., 1991; Li et al., 1993; Stanford et al., 1993). GS1 isoforms are responsible for assimilating the ammonium produced by the reduction of nitrate in roots, and are responsible for synthesizing Gln for the transport of nitrogen out of coty- ledons during seed germination. The major function of the GS2 isoform is to detoxify and reassimilate the ammonium released by photorespiration in leaves. Together, the GS enzymes play a central role in the assimilation of ammonium and the regulation of nitrogen metabolism in plants.

In the unicellular green alga Chlamydomonas reinhardtii GS is also the major enzyme for ammonium assimilation, which occurs exclusively via the GS-GOGAT cycle (Culli- more and Sims 1981b, 1981c; Stanford et al., 1993). Two isoforms of GS have been identified in C. reinhardtii, with enzymatic properties resembling those of GS1 and GS2 in higher plants (Florencio and Vega, 1983).

Physiological studies have shown that GS activity in C. reinhardtii goes through rapid, reversible deactivation depending on the nitrogen source and light conditions (Cul- limore, 1981). For example, approximately 70% of the GS activity was lost within 5 min when cells grown in nitrate were treated with 5 mM ammonium and switched to darkness. The activity of the deactivated enzyme could be partially restored in vivo by reilluminating the cell culture. Moreover, the repressive effect of ammonium on the up- take of nitrate and the activity and synthesis of nitrate

Abbreviations: GOGAT, glutamate synthase; GS, Gln synthetase; TAP, Tris-acetate-phosphate.

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988 Chen and Silflow Plant Physiol. Vol. 112, 1996

reductase was relaxed when the GS activity in the cell was deactivated by Met sulfoximine (Cullimore and Sims, 1981b). Based on this evidence, it was proposed that GS might also play a role in the control of nitrate assimilation (Cullimore and Sims, 1981b).

Regulation of GS is tightly controlled by various complex mechanisms operating at the pre- and posttranslational levels and at the level of enzyme activity. In higher plants GS activity is regulated at each of these levels (McNally et al., 1983; Chen and Kennedy, 1985; Tingey et al., 1987,1988; Edwards and Coruzzi, 1989; Kozaki et al., 1991; Hoelzle et al., 1992; Stanford et al., 1993); however, photosynthetic microorganisms appear to regulate GS activity mostly at the enzymatic and protein levels. In bacteria, the best- characterized posttranslational regulation consists of adenylation/ deadenylation of GS (Stadtman et al., 1970; Alef and Zumft, 1981; Nordlund et al., 1985). In cyanobacteria and green algae it has been proposed that allosteric regulation by products of Gln metabolism plays an important role at the enzymatic level (Florencio and Vega, 1983; Beudeker and Tabita, 1985; Merida et al., 1991). Because GS genes have not been isolated previously from green algae, gene regulation at the pretranslational level in these systems has not been reported.

To investigate the function of GS in nitrogen assimilation and the regulation of GS gene expression in algae, we isolated and characterized cDNA clones encoding GS from C. reinkardtii. The cDNAs and their derived amino acid sequences have extensive homology with those from higher plants. Our analysis indicated that GS1 is encoded by a single gene. Although two genomic sequences hybridize to GS2 probes, we found evidence for expression of only one GS2 gene. The expression of GS genes in cells growing in the presence of different nitrogen and carbon sources and different light conditions was investigated. The results indicate that expression of GS1 and GS2 genes is regulated differently from GS homologs in higher plants and differently from each other, reflecting their unique biological roles in C. reinhardtii. In addition, ammonium assimilation appears to be regulated by a different pathway than nitrate assimilation.

MATERIALS A N D METHODS

Chlamydomonas reinhardtii Cul t u re

For genomic DNA isolation, wild-type Cklamydomonas reinhardtii cells were grown in TAP liquid medium (Gor- man and Levine, 1965) bubbled with air at 25°C under continuous light. Unless specified differently, strain 826 (srl, mt') or strain A54 (ac17, srl, mt') was used as the wild type for RNA isolation (Schnell and Lefebvre, 1993). The growth conditions varied for different experiments and are detailed in "Results" and in the figure legends. For TAP/ NO, medium, NH,C1 was replaced with KNO, at the same molarity (7.5 mM). For TAP/acetate, acetic acid was omit- ted from the medium and the pH of the medium was adjusted with HC1. Other strains used for RNA isolation were A54-el8 (ac17, sul, nitl-Al, mt+), A54-g2 (ac17, srl,

nit24, mt+), A54-a29 (ac17, srl, nit8, mtt ; obtained from R. Schnell) and C49B (farl-1, mf+; obtained from D. Zhang).

lsolation of cDNA Clones

A C. reinhardtii cDNA library of poly(A) RNA isolated from the wild-type strain A55 (ac17, srl, mt-) was constructed in the AEXlox vector (Novagen, Madison, WI). The library was screened with maize GS1 or GS2 cDNAs at reduced hybridization stringency in 6X SSPE ( 1 X SSPE = 0.15 M NaCI, 0.01 M NaH,PO,, and mM EDTA, pH 7.4), 1 X Denhardt's solution, 50 mg/mL salmon sperm DNA, and 0.5% SDS at 60°C. Radioactive probes were prepared with [c I -~*P]~CTP using the random-primer method (Feinberg and Vogelstein, 1983). As necessary, cDNAs from positive clones were subcloned into pUC118 or pUC119 phagemids (Vieira and Messing, 1987).

D N A Sequence Determination and Sequence Analysis

DNA sequences were obtained using Sequenase 2.0 (United States Biochemical) with single-stranded tem- plates. The complete nucleotide sequence was determined for both DNA strands, and DNA and protein sequence analyses were performed with the Sequence Analysis Soft- ware Package (Genetics Computer Group, Madison, WI) (Devereux et al., 1984).

D N A lsolation and D N A Hybridizations

C. reinhardtii total genomic DNA was isolated according to Schnell and Lefebvre (1993). DNA was digested with restriction enzymes, electrophoresed on 0.9% agarose gels, and blotted to nylon membranes (Magna NT, Micron Sep- arations, Westborough, MA) following protocols supplied by the manufacturer. Probes for hybridization were cDNA fragments purified by agarose gel electrophoresis and the GeneClean I1 kit (BIO 101, La Jolla, CA). For GS1 it was a SalI-ApaI fragment (bp 167-630 in Fig. lA), and for GS2 the probe was an EcoRI-ApaI fragment (bp 1-552 in Fig 1B). Blots were hybridized with these probes labeled with [ C I - ~ ~ P ] ~ C T P by the random-primer method (Feinberg and Vogelstein, 1983). Hybridization was performed according to the manufacturer's protocol. After hybridization blots were washed twice with 2X SSPE/0.1% SDS for 10 min at room temperature, followed by two washes with 0.2 x SSPE/0.1% SDS for 30 min at 65°C.

RNA lsolation and Hybridizations

Total RNA was isolated from cultures of C. reinhardtii with a density of 2 X 106 to 4 X 106 cells/mL, as described previously (Schloss et al., 1984). Approximately 25 mg of total RNA was fractionated on each lane of 1.2% agarose gels in formaldehyde (Sambrook et al., 1989). The RNA was transferred to nylon membranes (Hybond N+, Amersham) and hybridized with DNA probes labeled with [ C I - ~ ~ P ] ~ C T P using the random-primer method (Feinberg and Vogelstein, 1983). The probe for GS1 transcripts was a 0.483-kb StyI-SacI fragment (3' noncoding region, bp 1332- 1815 in Fig. 1A). The probe for GS2 transcripts was a

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Gln Synthetase Genes in Chlamydomonas reinhardtii 989

0.503-kb SalI-HindIII fragment located at the 3' noncoding region of the cDNA (bp 1101-1604 in Fig. 1B). Hybridiza- tion reactions were carried out using protocols supplied by the manufacturer (Amersham). Each experiment was re- peated twice. The abundance of transcripts was quantified by determining the radioactivity of the specific hybridizing bands on the RNA blots with a radioanalytical imaging system (Ambis, San Diego, CA).

RESULTS

ldentification of GS cDNA Clones

Putative GS cDNAs were isolated from a C. reinhardtii cDNA library using maize cytosolic GS1 and chloroplastic GS2 cDNAs (Snustad et al., 1988; Li et al., 1993) as hybridization probes. When these clones were analyzed by restriction enzyme digestion, all of the cDNAs identified by the GS1 probe showed overlapping digestion patterns, as did all of the cDNA inserts identified by the GS2 probe. Thirty cDNA clones contained inserts between 1.6 and 2 kb, comparable in size to those of the GS cDNA clones of higher plants and large enough to include the entire coding sequence. Two cDNA clones, one selected with the maize GS1 clone and one with the maize GS2 clone, contained the largest cDNA inserts and were selected for DNA sequencing. In the rest of the article, clone GC581 is referred to as GS1 and clone GP1121 is referred to as GS2.

D N A Sequence Analysis

Figure 1 shows the entire nucleotide sequence of C. reinkardtii GS1 (A) and GS2 (8) cDNAs and their deduced amino acid sequences. The GS1 cDNA insert contains a coding sequence of 1146 bp. It has features described for other C. reinkurdtii cDNA clones, such as a high GC content (62%) and a characteristic putative polyadenylation signal, TGTAA (Silflow et al., 1985). It contains a long (800 bp) 3' noncoding sequence and a 5' noncoding sequence of 83 bp. The GS1 clone encodes a polypeptide of 382 amino acids

with a predicted molecular mass of 42 kD, which is comparable to the size of GS1 from other organisms. The GS2 cDNA has an open reading frame of 1140 bp. Because the GS2 cDNA contained only a short 5' noncoding sequence, a genomic fragment corresponding to this region was isolated and sequenced to attempt to determine whether the cDNA clone was complete. The genomic sequence contained an in-frame stop codon 117 bp upstream from the first ATG codon, confirming the authenticity of the start codon shown in Figure 18. The GS2 sequence also had a 62% GC content and the TGTAA polyadenylation signal, and encoded a polypeptide of 380 amino acids with a predicted molecular mass of 42 kD. The first 34 amino acids have the characteristics of a transit peptide of C. reinkardtii and higher plants, including a high content of hydroxy- lated and basic amino acids, no acidic residues, and Ala as the second residue, suggesting that the GS2 protein is localized in the chloroplast (Keegstra and Olsen, 1989; Franzén et al., 1990). The coding sequences for both GS1 and GS2 demonstrate a highly biased codon usage typical of most C. reinkardtii genes.

Sequence Comparison of C. reinhardtii GS with GS from Other Organisms

To understand the relationship of C. reinkardtii GS1 to GS1 enzymes from other organisms, we compared the deduced amino acid sequence of C. reinhardtii GS1 with those from six divergent organisms (Fig. 2). The comparison begins with the pairwise alignment of the two most similar sequences; other similar sequences are also posi- tioned closest to each other in the figure. The results indicated that C. reinkardtii GSl shares the highest homology with GS1 from pea and maize (74% similarity), supporting biochemical data suggesting that C. reinkardtii GS enzymes are structurally similar to those of higher plants (Florencio and Vega, 1983). The most striking unique feature of the C. veinkardtii GS1 enzyme is a 10-amino-acid insertion (residues at positions 127 to 136 in Fig. 2) relative to the pea and

. ' ..

. .. . .

Figure 1. Nucleotide and deduced amino acid sequences of the CS cDNAs encoding cytosolic GSI (A) and chloroplastic GS2 (6) of C. reinhardtii. Nucleotide sequences are shown in low- ercase letters; amino acid sequences (single- letter code) are shown in uppercase letters. The putative transit peptide in the CS2 sequence is underlined.



Chen and Silflow Plant Physiol. Vol. 1 1 2, 1996

1 O

M A A G E V G Y P A T D E K I O S L

Figure 2. Sequence comparison of C. rein!

140

P F Q Y K N . A I D N H I O E P M D K N

480

E A I D A Y I h L

rdtii GS1 enzymes from maize, pea, human, yeast, and S. typhimurium. The alignment was performed using the PileUp program in the GCG software package. Dots indicate gaps introduced to maximize sequence similarity. Amino acid residues identical in at least four species are indicated hy shading. The sequences were from the following sources: maize (Sakakihara et al., 1992), pea (Tingey et al., 1988), human (Gihhs et al., 1987), yeast (Magasanik and Minehart, 1992), and S. typhimurium (Almassy et al., 1986). Sequences within the active site include amino acid residues at positions 82 to 99, 254 to 263, 297 to 312, and 384 to 390 (Eisenherg et al., 1987).

maize sequences. The comparison also shows that the four regions making up the active site of the Salmonella typhimurium GS enzyme (Almassy et al., 1986; Eisenberg et al., 1987) are highly conserved in a11 six species, suggesting that C. reinhardtii GS1 has the same active site as GS1 from other organisms.

Figure 3 shows the comparison between C. reinhardtii, maize, and pea GS2 amino acid sequences. The amino- terminal regions, which correspond to the putative transit peptides, are very divergent, as is typical for transit peptides in other nuclear-encoded proteins in the chloroplast (Demmin et al., 1989; Chen and Vierling, 1991). The putative transit peptide for C. reinhardtii GS2 was shorter (34 amino acids) than those of maize and pea (42 and 50 amino acids). This observation is consistent with the structure of other C. reinhardtii chloroplast transit peptides (mean length, 30 amino acids), which are generally shorter than those of higher plants (mean length, 60 amino acids; Franzén et al., 1990). In the region representing the mature protein, C. reinhardtii GS2 was 70% similar to pea and

maize GS2. The unique characteristics of the C. reinhardtii GS2 enzyme include a 9-amino-acid insertion (residues at positions 90-98 in Fig. 3) relative to the pea and maize sequence and the lack of a carboxyl-terminal extension of 16 residues found in GS2 enzymes of higher plants (Snus- tad et al., 1988). It has been proposed that chloroplasts of C. reinhardtii and higher plants are of different evolutionary origin (Franzén et al., 1990). The divergence of the GS2 sequence of C. reinhardtii from higher plants at both the amino and the carboxyl termini would be consistent with the speculation that GS2 enzymes in C. reinhardtii and higher plants evolved independently.

Genomic D N A Blot Analysis

To determine the number of genes encoding GS in C. reinhardtii, genomic DNA blot analysis was performed. As shown in Figure 4, one hybridizing fragment was detected using the GS1 probe for a11 of the enzymes tested, indicating that there is a single gene for GS1 in C. reinhardtii. For GS2, two hybridizing fragments of similar intensity were detected consistently using either a 5' or a 3' coding region of the cDNA as a probe. However, when the 3' noncoding fragment was used as a gene-specific probe, only one of the two fragments hybridized with the probe (data not shown). These results indicated that there were probably two genomic sequences coding for GS2, and these were termed GS2-1 and GS2-2.

To determine whether transcripts for both GS2 genes were present, the 3' noncoding region of 10 independent GS2 cDNA clones (as indicated by their unique poly[A] tail lengths) were sequenced. Comparison of the sequences indicated that a11 of these GS2 cDNA clones are derived .from the GS2-1 gene.

GS Expression under Different Nitrogen Sources

The synthesis and biological activity of almost a11 of the components in the nitrate assimilation pathway in C. rein-

170 zw

Figure 3. Comparison of deduced amino acid sequences of the GS2 cDNA of C. rernhardtrr with GS2 from maize and pea The comparison was performed using the PileUp program in the GCG software package Amino acid residues identical in at least two species are presented hy shading The maize sequence is from Snustad et al (1 988) and the pea sequence is from Tingey et al. (1 988).



Gin Synthetase Genes in Chlamydomonas reinhardtii 991

B

S 3 O = 1M > a a Ea. a. u> < / > < / > = oi > « « ETO. a c/> co to

Figure 4. Genomic DNA blot analysis. Genomic DNA (2 /xg/lane)was digested with restriction enzymes H/ndlll, Psfl, Pvull, Sacl, 5a/l,or Smal. The digested DNA was then fractionated on a 0.9% agarosegel and transferred to a nylon membrane. The DNA was hybridizedwith a radioactive probe of the 5' coding region of the C. reinhardtiiGS1 cDNA (A) or the 5' coding region of the GS2 cDNA (B).

hardtii are tightly regulated by the nitrogen source (Fernan-dez and Cardenas, 1989). Previous studies indicate that thenitrogen source also regulates GS activities in this system(Cullimore, 1981). To understand whether the regulation isoccurring at the pre- or posttranslational level, we exam-ined the expression of GS mRNAs under different nitrogenconditions.

C. reinhardtii cells were grown in TAP liquid mediumwith ammonium as the only nitrogen source for 2 to 3 d toa density of 2 X 106 to 4 X 106 cells/mL. The cells were thendivided into aliquots, washed, and resuspended in TAP (0time point) or TAP/NO3 medium with nitrate as the nitro-gen source for 30, 60, or 75 min. Total RNA was isolatedand analyzed by RNA gel blot analysis (Fig. 5A). GS1transcripts were detectable when cells were grown in me-dium containing ammonium, but the GS1 transcript levelwas dramatically increased when the cells were switchedto nitrate medium. Radioactive signals on the RNA blotswere quantified by radioanalytical imaging, averaged be-tween experiments, and normalized to the levels of theribosomal protein S14 (Cry-1) transcript (Fig. 5B). Within60 to 75 min the level of GS1 transcripts increased approx-imately 5-fold, indicating that ammonium represses GS1transcript levels, that nitrate induces GS1 transcript levels,or that a combination of both occurs. In further experi-ments in which cells were switched from medium withammonium to medium with no nitrogen, the GS1 tran-script levels also increased but not to the level observed inthe presence of nitrate (data not shown). These resultsconfirm that ammonium represses GS1 transcription ordestabilizes GS1 transcripts, and that nitrate induces GS1transcription or stabilizes its transcripts.

Because the genomic blot analysis indicated that the C.reinhardtii genome contains two sequences encoding GS2, a

BTAP/NOs

TAP o o mto <o r^

GS1

GS2

Cry-1

30 60time (min)

75

scrip

t abu

ndan

ceU

J -fa

U

i C

S 20)

Js 105a:

GS2

Figure 5. Effect of ammonium and nitrate on GStranscript levels. Cells (strain A54) were grownin TAP liquid medium with ammonium as theonly nitrogen source for 2 to 3 d under constantlight. The cells were then washed and trans-ferred to TAP/NO3 medium with nitrate as thenitrogen source for 30, 60, or 75 min. Total RNAwas isolated, fractionated by gel electrophoresis(25 /xg/lane), and blotted to membranes. TheRNA blots were hybridized with GS1 or GS2gene-specific cDNA probes as indicated. Theblots were also hybridized with the ribosomalprotein SI 4 (Cry-1) cDNA probe to show equalloading (Nelson et al., 1994). A shows a repre-sentative autoradiograph. The mean levels andSE values of GS transcripts are shown in graphsin B. The mean level of GS transcripts is calcu-lated from data of at least two separate experi-ments and is normalized to the Cry-1 transcriptlevel.

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992 Chen and Silflow Plant Physiol. Vol. 11 2, 1996

gene-specific probe containing only the 3' noncoding region of GS2-1 was used for RNA blot analysis. The gene- specificity of the probe was confirmed by its hybridizing with only one of the two fragments on the genomic DNA blot (data not shown). Sequencing analysis of the GS2-1 and GS2-2 genomic clones also indicated that they have different 3' noncoding sequences (data not shown). The hybridization pattern for GS2 transcripts was different from that of GS1 transcripts (Fig. 5B); a similar level of GS2 transcripts was detected in cells grown on ammonium medium or transferred to nitrate medium.

GS Expression under Different Light Conditions

To determine whether light also plays a regulatory role in GS gene expression, C. reinkardfii cells were grown on TAP medium in total darkness for 5 d and then exposed to white light for 3, 8, or 48 h. Total RNA was isolated, fractionated by gel electrophoresis, transferred to RNA blots, and hybridized with GS1 or GS2 gene-specific probes. GS1 transcripts (Fig. 6A) were present when the cells were grown in the dark. When the cells were exposed to light, the GS1 transcript level increased within 3 h, reaching a level 2- to 3-fold higher than that present in the dark-grown cells at 8 h. During further exposure to light GS1 transcript levels decreased, which may indicate a de- crease in the absolute amount of GS1 mRNA, or may reflect the diminished proportion of GS1 mRNAs relative to the increasing number of transcripts for light-responsive genes. For GS2 (Fig. 6B), the transcript level was not affected within the first 8 h after exposure to light. After 48 h, it increased approximately 1.5-fold.

GS Expression under Different CO, Conditions

The effect of light on GS expression suggests that GS transcript levels may depend on the metabolic status of chloroplasts. An important factor affecting chloroplast metabolism is the CO, level. To determine the effect of CO, on the expression of GS genes, cells were grown mixtrophically in TAP medium or phototrophically in TAPIacetate

B

lime (h) time (h)

Figure 6. GS expression is regulated by light. Cells (strain 826) were grown in TAP medium for 5 d in total darkness. The cells were then exposed to white light for 3 , 8, or 48 h. Total RNA was isolated, blotted to membranes, and hybridized with GSI (A) or GS2 (B) gene-specific cDNA probes. The levels of GS transcripts on the blots were quantified with radioanalytical imaging, normalized to Cry-1 transcript level, and averaged between at least two experiments. The error bars indicate SE.

A B I I 10, I

8 2.0 T

Figure 7. Effect of CO, on GS expression. Cells (strain 826) were grown mixtrophically in TAP medium or phototrophically in TAP/ acetate (AC) medium and bubbled with either air or 5% CO, for 3 d as indicated. Total RNA was isolated and analyzed on blots hybridized with GSI (A) or GS2 (B) gene-specific cDNA probes. The level of GS transcripts on the blots was quantified, normalized to Cry-I, and averaged between two separate experiments.

medium and bubbled with air or 5% CO, for 3 d. Total RNA from these cells was isolated and analyzed on RNA gel blots. For both GS1 (Fig. 7A) and GS2 (Fig. 7B) cells grown in CO, had only slightly higher levels of transcripts.

GS Expression in Mutants with Defects in Regulation of Nitrogen Metabolism

Physiological studies have suggested that GS may participate in the control of nitrate assimilation (Cullimore and Sims, 1981b). In funga1 systems, some regulatory factors of the nitrate assimilation pathway, such as the positive regulator Nit2 in Neurospora, also extend their regulatory function to ammonium assimilation and 1 or amino acid metabolism (Marzluf, 1981). Because severa1 genes in the nitrate assimilation pathway, including regulatory factors, have been identified in C. reinhardtii it was possible to examine the role of these genes in ammonium assimilation. The regulation of GS gene expression was examined in mutants with defects in the nitrate assimilation pathway, including the nitrate reductase gene nifl (Fernández et al., 1989) and the positive regulatory gene nit2 (Schnell and Lefebvre, 1993). Cells containing the nif8 or the farl mutation were also analyzed. Although the function of the NIT8 gene is unknown, cells containing the nit8 mutations are unable to utilize nitrate at a11 or utilize nitrite only poorly (Wang, 1995). Cells containing the furl mutation, unlike wild-type cells, produce nitrate reductase even in the presence of ammonium, suggesting that the FARl gene is a negative regulatory gene (Zhang and Lefebvre, 1996).

Mutant cells were grown in TAP liquid medium with ammonium as the only nitrogen source for 2 to 3 d. The cells were then transferred to TAPINO, medium with nitrate as the nitrogen source for 30, 60, or 75 min. Total RNA was isolated and underwent blot analysis. The results indicate that GS1 transcripts could be detected when mutant cells were grown in ammonium medium (Fig. 8). After transfer to nitrate medium, GS1 transcript levels in nitl, nit2, nit8, and furl mutants started to increase in a manner similar to that seen in wild-type cells. A similar analysis




6 0 "."

O 30

time (min)

75

Figure 8. GS1 transcript levels in cells with defects in the nitrate assimilation pathway. Cells containing the mutations nitl (nitrate reductase gene; Fernández et al., 1989), nit2 (positive regulatory gene; Schnell and Lefebvre, 1993), nit8 (gene function unknown, but mutants cannot utilize nitrite; Wang, 1995), and farl (putative negative regulatory gene; Zhang and Lefebvre, 1996) were grown in TAP liquid medium with ammonium as a nitrogen source for 2 to 3 d under constant light. The cells were transferred to TAP/NO, medium with nitrate as t h e nitrogen source for 30, 60, or 75 min. Total RNA was isolated, fractionated by gel electrophoresis, and blotted to membranes. The R N A was hybridized with a GS1 gene-specific cDNA probe. The levels of GS transcripts on the blots were quantified, normalized to Cry-1, and averaged between two separate experiments.

indicated that, as in wild-type cells, the GS2 transcript levels in these mutant cells were not affected by ammonium or nitrate (data not shown).

DISCUSSION

In nonleguminous higher plants, two isoforms of GS have been identified (reviewed by Last, 1993; Lam et al., 1995). In green algae, some species (including Euglena spp.) contain only one GS isoform that can be located in either the cytosol or the chloroplast. In contrast, other species (including C. reinhardtii) have two isoforms, one cytosolic and one chloroplastic (Florencio and Vega, 1983; Sumar et al., 1984; Casselton et al., 1986; Ahmad and Hellebust, 1987; Fischer and Klein, 1988). The enzymatic characteristics of the two GS isoenzymes in C. reinhardtii resemble those of GS1 and GS2 in higher plants (Florencio and Vega, 1983). Our isolation of two types of cDNAs further supports the suggestion that there are at least two isoforms of GS in C. reinkardtii. Together with the observation that two types of GOGAT (Fd-GOGAT and NAD[P]H-GOGAT) exist in C. reinkardtii (Cullimore and Sims, 1981c), our results indicate that two GS-GOGAT systems function in this species.

In all species examined to date, GS2 appears to be encoded by a single nuclear gene (Cullimore et al., 1984; Tingey et al., 1987; Peterman and Goodman, 1991; Li et al., 1993). The results from this study indicate that C. reinkardtii is unique in that it contains two GS2 sequences (GS2-1 and GS2-2), although no evidence was found for expression of

the GS2-2 sequence. It is possible that the GS2-2 cDNA may not be in the cDNA library screened for this study because its mRNA was not expressed under the growth conditions used for the cells from which the library was constructed. To determine whether the GS2 expression patterns were due primarily to the GS2-1 gene, the RNA gel blots used for the experiments shown in Figures 5, 6, and 7 were hybridized with the entire coding sequence of the GS2-1 cDNA, which would hybridize with both GS2-1 and GS2-2 transcripts. In each case, the pattern of gene expression was the same as that obtained with the GS2-1 gene- specific probe (data not shown). Results from these experiments, which covered many different culture conditions, suggested that the GS2-2 gene was not expressed or was expressed at a low level, and had an expression pattern similar to that of the GS2-1 gene.

Because GS is a key enzyme in nitrogen assimilation, its synthesis and activity are under tight regulation by cellular metabolism and environmental signals. In higher plants, GS expression is regulated by environmental signals such as light, nitrate, and ammonium, and by the metabolic status of the cell. The response of GS activity and gene expression to environmental ammonium or nitrate varies depending on the specific member of the gene family and the plant species examined (Hirel et al., 1987; Kozaki et al., 1991; Hoelzle et al., 1992; Stanford et al., 1993; Sukanya et al., 1994). However, when the genes are responsive, ammonium and nitrate always up-regulate GS gene expression and/or GS activity.

In C. reinkardtii, components in the nitrate assimilation pathway are tightly regulated by nitrogen sources, as are GS enzyme activities. However, ammonium seems to have an opposite effect on GS activity in green algae than it does in higher plants. Instead of up-regulating GS activity, its presence in the environment represses it in C. reinkardtii. For example, within 5 min approximately 70% of the GS activity was deactivated when cells grown in nitrate were treated with 5 mM ammonium (Cullimore, 1981). This re- port provides the first evidence, to our knowledge, that ammonium and nitrate also regulate GS gene expression at the transcript level in C. reinkardtii. The presence of ammonium in the medium represses the transcription of the GS1 gene or destabilizes its mRNA, whereas nitrate induces GS1 transcription or increases the stability of its transcript. Our results are also consistent with the observation that the relative ratio of GS2 to GS1 activity increases and the total GS activity decreases when cells are transferred from nitrate to ammonium (Fischer and Klein, 1988).

Repression of GS synthesis by ammonium has been observed in photosynthetic bacteria and in cyanobacteria. In cyanobacteria ammonium itself seems to be the corepressor of GS synthesis (Orr and Haselkorn, 1982), whereas in photosynthetic bacteria Gln, or a metabolic product of Gln, appears to be responsible for eliciting the repression (Ro- mero et al., 1985). Whether ammonium itself or its metab- olites are direct corepressors for the GS enzyme or for GS gene expression is still unclear in green algae (Cullimore and Sims, 1981b). In the green alga Monoraphidium braunii it has been suggested that Gln, not ammonium, is the direct



994 Chen and Silflow Plant Physiol. Vol. 1 1 2, 1996

negative regulator of GS synthesis at the protein level (García-Fernández et al., 1995). In C. reinhardtii, however, an increase in GS activity was observed after Gln addition to cells (Florencio and Vega, 1983). In C. reinhardtii, L-Gln does not seem to be efficiently transported into the cell, which might result in nitrogen starvation when it is supplied as the only nitrogen source (Cullimore and Sims, 1981b; Menacho and Vega, 1989). This could explain the above-mentioned increase in GS activity, because in green algae the absence of nitrogenous compounds produces a general increase in enzymes of the GS-GOGAT cycle (Cul- limore and Sims, 1981b; Paul and Cooksey, 1981). The repression of GS transcription and GS activity by ammonium or its metabolic products may serve to conserve ATP, reductant, and carbon skeletons under conditions at which the supply of these compounds is limited and ammonium is in excess.

Light regulates GS gene expression and GS activity in higher plants (Tingey et al., 1987, 1988; Edwards and Coruzzi, 1989). In Phaseolus vulgaris the GS2 mRNA level increased over 10-fold in response to illumination (Cock et al., 1991). Expression of a chimeric gene containing a pea GS2 promoter and the GUS reporter gene construct in transgenic tobacco revealed that the promoter contains a cis-acting DNA element involved in light regulation (Ed- wards et al., 1990). Light also regulates GS enzyme activity in C. reinhardtii. GS activity underwent reversible deactivation when C. reinhardtii cells were switched from light to dark (Cullimore, 1981). Our results demonstrate that light also regulates GS expression at the level of transcript accumulation. GS1 transcript levels increased within 3 h after exposure of cells to light, suggesting that it is regulated by light directly. In contrast to GS1, the increase of C. reinhardtii GS2 transcripts could be detected only after 48 h of light exposure, suggesting that unlike GS2 in higher plants, GS2 transcript levels in C. reinhardtii are regulated by the light-induced metabolic changes in chloroplasts. In addition to providing energy for photosynthesis, leading to the accumulation of carbon skeletons required for ammonium assimilation, light also induces many changes in chloroplast and cellular metabolism. The induction of GS2 gene expression in pea has been shown to result partly from the light-induced changes in chloroplast metabolism, in addition to the action of phytochrome (Edwards and Coruzzi, 1989). This indirect effect is slower because it depends on light-induced events and/ or requires factors associated with the presence of mature chloroplasts, which are not present in dark-grown cells (Edwards and Coruzzi, 1989). Even though wild-type C. reinhardtii cells can produce a fully developed photosynthetic system in the dark (Malmoe et al., 1988), the induction of GS2 may still require other light-induced changes in chloroplasts. Phy- tochrome is thought to be the primary light receptor for mediating GS expression in higher plants (Tingey et al., 1988; Edwards and Coruzzi, 1989; Sakamoto et al., 1990), but it has not been identified in C. reinhardtii or other green algae (Bonenberger et al., 1994). Other receptors, such as blue light photoreceptors, may play a role in the response of GS gene expression to light. The lack of

phytochrome in C. reinhardtii also may explain why the increase in GS2 expression in response to light is rela- tively slow.

The effect of light on GS gene expression suggests that GS gene transcript levels depend on the metabolic status of chloroplasts. In higher plants, one of the major functions of GS2 is to detoxify the ammonium released by photorespiration. Studies of pea GS2 mRNA accumulation under different photorespiration conditions demonstrated that plants maintain higher levels of GS2 when they are grown under photorespiratory conditions (Edwards and Coruzzi, 1989). It has been proposed that GS2 expression is induced by ammonium released by photorespiration (Edwards and Coruzzi, 1989). However, in a study of P. vulgaris GS2 gene expression the increase in GS2 mRNA under photorespiration growth conditions was not directly regulated by the flux of photorespiratory ammonium, but rather was probably the result of indirect, long-term effects on cellular metabolism such as differential growth rate or differences in the cellular pH (Cock et al., 1991). In C. reinhardtii photorespiration is limited due to the C0,-concentrating system (Lloyd et al., 1977; Birmingham and Coleman, 1979; Coleman and Coleman, 1980; Cullimore and Sims, 1980). However, the presence of a C0,-concentrating system does not appear to repress the photorespiratory pathway (Marek and Spalding, 1991; Ramazanov and Cardenas, 1992, 1994). It has been shown that C. reinhardtii cells increase their ammonium excretion in the presence of Met sulfoximine when transferred from 5% CO, to low CO, (air) growth conditions (Ramazanov and Cardenas, 1992, 1994). Furthermore, C. reinhardtii GS2 enzyme activity iso-

higher than that isolated from cells grown in 5% CO,, whereas GS1 enzyme activity remained the same in cells grown under both conditions (Ramazanov and Cardenas, 1994). In the present study, slightly higher levels of GS2 transcripts were detected in cells grown with 5% CO, compared with cells grown in air, suggesting that the decreased activity of GS2 observed in cells grown in 5% CO, is not regulated at the level of transcript abundance.

Based on physiological studies, Cullimore and Sims (1981b) have proposed that GS may participate in the control of nitrate assimilation in C. reinhardtii. Some factors that regulate the nitrate assimilation pathway in funga1 systems, such as Nit2 in Neurospora, also function in the regulation of ammonium assimilation and / or amino acid metabolism (Marzluf, 1981). In the current study, we investigated a possible role for the regulatory components of the nitrate assimilation pathway in ammonium assimilation in C. reinhardtii. Our results indicate that, unlike in Neurospora, in C. reinhardtii the positive regulatory gene, NIT2, and the negative regulatory gene, FAX1, do not function in the regulation of ammonium assimilation. Therefore, ammonium assimilation and nitrate assimilation are probably regulated by different mechanisms in C. reinhardfii.

Because of the amenability of C. reinhardtii to genetic and molecular analyses, the results from this study will enable us to further investigate the role of GS in nitrogen metab-

lated from chloroplasts of cells grown in low CO, was 30%




olism and to define the regulatory elements and factors for ammonium assimilation. For example, genetic experiments i n C. veinhardtii have led to the identification of the only known nitrate regulatory gene (NIT2) in photosynthetic eukaryotes (Schnell and Lefebvre, 1993; Crawford, 1995). The cloning of GS genes also provides candidate genes for creating dominant selectable markers for C. reinhardtii transformation.

ACKNOWLEDCMENTS

We thank Dr. D. Peter Snustad for the generous gift of the maize GS1 and GS2 cDNAs, and Dr. Carro11 P. Vance (U.S. Department of Agriculture, Agricultura1 Research Service) for providing the Am- bis radioanalytical imaging system. We also thank Drs. Paul A. Lefebvre and D. Peter Snustad for helpful advice regarding the manuscript.

Received January 24, 1996; accepted July 9, 1996. Copyright Clearance Center: 0032-0889/96/ 112/0987/ 10. The GenBank accession numbers for the sequences described in

this article are U46207 (GSI) and U46208 (GSZ).

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Documents

lsolation and Characterization of Clutamine …et al., 1993). ln higher plants, the genes encoding individual GS isoforms are regulated differently by environmental signals such as