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M2 seed screening for nitrate reductase deficiency in Nicotiana plumbaginifolia
Plant Science. 76 (1991) 109 114 Elsevier Scientific Publishers Ireland Ltd.
109
M2 seed screening for nitrate reductase deficiency in Nicotiana
plumbaginifolia F r 6 d 6 r i q u e Pelsy, J o c e l y n e K r o n e n b e r g e r , J e a n - M a r i e Pollien a n d M i c h e l C a b o c h e Laboratoire de Biologie Cellulaire, INRA-Versailles 78026, Versailles Cedex (France)
Received December 10th, 1990; revision received February 1st, 1991; accepted February Ist, 1991) Nitrate reductase-deficient mutants can be isolated from protoplast-derivcd cells on thc basis of chlorate resistance. Since nitrate assimilation is a highly regulated and tissue specific process, it was expected that mutant selection at the phmt level could lead to the identification of new genes involved in the control of nitrate assimilation. Three selective procedures were compared for the selection of mutants impaired in nitrate assimilation, from M2 seeds: chlorate selection, and rescue of plants specifically unable to grow on low, or high, nitrate concentrations. The rescue of some of the plantlets unable to grow on nitratc was z,chicved through a shoot culture step by treatment with growth regulators. All three procedures led to comparablc frcqucncics of around 5 x 10-3 in the isolation of mutants totally impaired for nitrate assimilation. The majority of these mutants were of the cnx typc, as opposed to mutants selected for chlorate resistance at the cell level, which are predominantly of the tlio typc. ,ia mutants and mutants with leaky phenotypes were also identified, but unexpectedly no other class of mutants totally impaired in nitrate assimilation was identified among isolated mutants. The implications of these data are that M2 seed screening can be used as an alternative route to mutant selection at the cell level for the isolation of mutants totally impaired in nitrate assimilation. This approach can be cnvisagcd for the isolation of nitrate reductase-deficient mutants from species recalcitrant to in vitro manipulation at the cell level and subsequent regeneration into fertile plants. Key words." mutant; selection; chlorate; nitrate assimilation; Nicotiana plumbaginifolia
Introduction
The generation, screening and analysis of mutants is a powerful tool used extensively in the analysis of plant functions. Nitrate reductase-deficient mutants (NR-) have been selected to study the first step of the nitrate assimilation pathway in higher plants. Mutants can be selected from mutagenized cultures of protoplast-derived cells on the basis of chlorateresistance and regenerated into plants. N R mutants have been selected from cell cultures of Nicotiana tabacum and N. plumbaginifolia [1,2] for resistance tO chlorate. These mutants are fully deficient for nitrate assimilation. Alternatively, mutants can be obtained by screening second generation mutagen-treated (M2) seedlings for chlorate Correspondence to: Fr6d6rique Pelsy, Laboratoire de Biologic Cellulaire, INRA-Versailles, 78026 Versailles, France.
r e s i s t a n c e , o r f o r loss o f t h e e n z y m e a c t i v i t y . F o r instance, Arabidopsis, barley and pea chlorateresistant mutants have been isolated from M2 seedlings [3--5]. NADH or NADPH-dependant nitrate reductase-deficient mutant plants of barley and pea have been identified by in vivo NR activity a s s a y o n l e a f f r a g m e n t s [5]. T h e s e p r o c e d u r e s directly lead to the isolation of mutant plants w h i c h a r e u s u a l l y fertile, a n d o v e r c o m e t h e p r o b l e m s w h i c h s o m e t i m e s e x i s t in t h e r e g e n e r a t i o n o f plants from mutant cell lines. S i n c e n i t r a t e a s s i m i l a t i o n is a h i g h l y r e g u l a t e d a n d t i s s u e s p e c i f i c p r o c e s s e d , it w a s e x p e c t e d t h a t m u t a n t s e l e c t i o n a t t h e p l a n t level c o u l d l e a d t o t h e i s o l a tion of new classes of mutants. A drawback of the a p p r o a c h is t h a t m o s t o f t h e m u t a n t s i s o l a t e d b y these screening procedures are leaky, and t h e r e f o r e still a b l e t o g r o w o n n i t r a t e a s sole nitrogen source. This makes difficult the task of their biochemical analysis.
0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
110
Nitrate reductase-deficient mutants are usually classified on two criteria. A first classification is based on the ability of the mutant to retain a xanthine dehydrogenase ( X D H test) activity, an enzyme which shares with nitrate reductase the molybdenum containing cofactor (MoCo). Mutants showing X D H activity, are classified as apoenzyme-deficient mutants (nia mutants). In contrast, those lacking both N R and D H activities are classified as cofactor-deficient (cnx mutants), and must be defective in the biosynthesis or the processing of the MoCo. This biochemical characterization needs to be confirmed by somatic hybridization experiments [l,fr--8] and by genetic analysis to classify the mutants into separate complementation groups [9]. In our laboratory, a collection of 211 chlorate-resistant mutants of N. plumbaginifolia has been selected from protoplast cultures and characterized [2]. All of these were completely defective for the N R activity and carried a monogenic recessive mutation affecting structural genes. By the X D H test, 70 mutants were classed as cnx, which represent 33% of the collection. All fertile mutants were confirmed by genetic analysis and assigned to 7 complementation groups [2,10]: one nia group and 6 different cnx groups (cnx A to F). This paper describes the selection and characterization of nitrate reductase-deficient mutants isolated from M2 progeny of ethylmethane sulfonate (EMS) mutagenized seedlings and selected either for a deficiency in nitrate utilization or for their resistance to 30 mM chlorate. That auxotrophy for nitrate utilization can be used as a selective screen not only at the cell level, as previously shown for Hyoscyamus muticus [11], but also at the plant level is also demonstrated. This latter strategy has been improved with the aim of selecting new classes of mutants affected in the nitrate assimilation pathway. Materials and Methods
Mutagenic treatments Seeds of the haplo-diploidized N. plumbaginifolia cv. Viviani line P B H I were used throughout these experiments. The seeds were mutagenized by soaking in distilled water containing 0.4, 0.5 or
0.6% EMS for 16 h, then washed three times with distilled water, dried, and sown in the greenhouse. Developing plants were then potted individually and grown to maturity. M2 seeds were harvested in bulk from 24 mutagenized plants and surface sterilized.
Mutant selection Approximately 200~300 seeds from each of 118 batches were sown on modified medium B [2] containing 10 m M KNO3 as the sole nitrogen source. In parallel, chlorate selections were carried out on the same batches of seeds, under sterile conditions, on modified medium B containing 10 m M diammonium succinate as the sole nitrogen source, plus 30 m M potassium chlorate. Germinations were carried out in a controlled culture r o o m [10]. After germination on nitrate, NR-deficient seedlings turned white and stopped growing at the cotyledon stage of development. Plantlets displaying this phenotype were rescued on a medium containing diammonium succinate (10 m M ) and KC1 (10 mM) and vegetatively multiplied. The putative chlorate-resistant clones were also subcultured on this ammonium-containing medium. Under selective conditions involving 10 m M nitrate as a selective screen, albino, pale-green, and lethal mutants were discarded on the basis of a similar phenotype on a nitrate- or on an ammonium-containing medium. A third series of selections was performed on 69 of the batches previously used for nitrate and chlorate selection. These selections were carried out on medium B containing 1 m M KNO3. After germination, non-growing plantlets were again rescued on the ammonium-containing medium. Under these selective conditions, plantlets unable to grow upon transfer to ammonium, could occasionally be rescued on the same medium with 0.5 #M naphthaleneacetic acid and 1 #M benzyladenine added. This treatment induced shoot formation, and the shoots obtained were rooted on a hormone-free medium containing a m m o n i u m as the nitrogen source. Plants were further confirmed to be genetically impaired in nitrate metabolism by progeny analysis. Since N R deficient mutants grow poorly on their own roots, putative mutants were routinely grafted onto N. tabacum cv.
111
Wisconsin-38 plants and grown in the greenhouse with a standard nutrient solution containing nitrate and ammonium [12]. Crosses were performed between flowering grafts and reference nia E23 or cnx mutant plants. Detection of X D H activity Young green leaves were collected either on plantlets grown in plastic pots or on grafted rosettes. X D H activity was detected in crude enzyme extract according to the method of Mendel and MUller [13] after electrophoretic separation in non-denaturing polyacrylamide gels and staining in situ for X D H activity. Results and Discussion
Optimization o f mutagenesis Since EMS mutagenesis experiments were not previously described on seeds of N. plumbaginifolia, a true diploid species, a dose-response study has been performed to establish optimal conditions. Three concentrations of EMS were compared with regard to their induction of albino sectors in M 1 plants, and to their remaining fertility. Table I shows that a concentration of 0.5% EMS led to the highest induction of mutated sectors, without much impairment of fertility (97%). Above this concentration, a rapid increase in the proportion of sterile plants was detected (20% at an EMS concentration of 0.6%). We assume that this increase is related to the generation of chromosome breaks and deletions, which are known to strongly reduce the production o f viable pollen. The optimal concentration for EMS (40
Table !. Chlorophyll deficiencies and sterility induced by three EMS treatments on seeds of N. plumbagin(]'olia Mutagenic treatment
Albino chimera (%)
Sterile plants (%)
None 0.4% EMS (32.2 mM) 0.5% EMS (40.25 m M ) 0.6% EMS (48.3 m M )
0.9 2.11 2.26
-0.01 0.03 0.20
mM) is comparable to the routinely used for mutagenesis of an other diploid species, Arabidopsis thaliana [4]. Selection and characterization of mutants resistant to chlorate As the selection for chlorate resistance usually leads to the recovery of mutants totally deficient in nitrate reductase, these mutants are counterselected, in greenhouse conditions, due to their requirement for an organic acid to utilize ammonium efficiently and to grow [2]. To be able to analyse all chlorate resistant plantlets, selections were therefore performed under aseptic conditions, on a medium allowing comparable growth of wild type and mutant plants on ammonium as the sole nitrogen source [2]. Twenty putative chlorate-resistant plantlets were isolated in independent pools of M2 seeds and the progeny of seventeen clones were further characterized (Table II). Among them ten mutants were confirmed to carry a nia or a cnx mutation conferring a total deficiency for nitrate assimilation (Table III). Three clones displaying low fertility were erroneously selected, as their progeny displayed a wild type phenotype. Three clones remain to be characterized. This experiment demonstrated that chlorate can be used to efficiently select mutants totally impaired in nitrate assimilation, from M2 seedlings, since more than 50% of the selected mutated lines were of this type. Selection and characterization of mutants unable to grow on 10 m M nitrate A selection based on the rescue of M2 plantlets unable to grow on nitrate was performed with the aim of increasing the number of loci accessible to genetic analysis. Since N. plumbaginifolia plantlets can be grown on nitrite as the sole nitrogen source, it was expected that, for instance, mutations impairing nitrite reductase activity, or nitrite transport into the chloroplast, could be identified by this approach. Among 53 lines initially selected for their inability to grow on nitrate, 17 turned out to be indistinguishable from the wild type (Table II). It was therefore assumed that their initially impaired growth on nitrate resulted of an independent physiological defect, possibly related to the
112 Table II. A summary of the three selection procedures for obtaining mutants deficient in nitrate assimilation. NS, not scored; NT, not tested. Selective conditions
Chlorate (30 raM)
Nitrate (10 raM)
Nitrate (1 mM)
No. of mutagenized plants tested No. of putative mutants selected Shoot cultures displaying the same phenotype on nitrate or ammonium: Albino, chlorotic or lethals Comparable to wild type Clones rescued on ammonium Clones rescued by regeneration of shoots on ammonium plus hormones Clones transferred to the greenhouse and progeny tested Progeny unable to grow on: Nitrate 10 mM Nitrate I mM Progeny segregationg lethal, albino or pale-green mutants Progeny with wild type phenotype Frequency of mutants defective in nitrate assimilation
2832 20
2832 53
1653 103
NT 17 36 NT
55 12 36 17 out of 55
-18 17
31
50
13 NT I
15 NT 2
7 9 7
3 45.9/10 000
14 52.9/10 000
improper maturation of these seeds during ripening. Two plants expressed a pale-green phenotype which was transmitted to their progeny. As this phenotype was expressed on nitrate as well as ammonium-containing medium, it was not therefore the consequence of a specific defect in nitrate assimilation. The remaining mutants all carried recessive mutations preventing growth on 10 mM nitrate. Ten of them have been classified as cnx on the basis of X D H deficiency, and four as nia by genetic crosses with nia E23. Mutant 200 N remains to be characterized. No mutant defective for nitrite reductase activity has been selected in this experiment. Selection and characterization o f mutants unable to grow on 1 m M nitrate
An other round of selection to isolate M2 plantlets unable to grow on nitrate was carried out with two modifications, on some of the batches of seeds previously used for chlorate and nitrate selection. Lower concentrations of 0.2 or 1 mM nitrate have been used: the lowest concentration
33 54.4/10 000
resulted in the rapid depletion of the nitrogen source from the culture medium, so that it was difficult to distinguish between nitrate depletion and nitrate utilization deficiency by eye screening. This screening was therefore routinely performed on 1 mM nitrate. This procedure again led to the identification of three mutants of the nia type and four mutants of the cnx type, some of these mutants being identified as expected in batches of M2 seeds known from previous selections to contain nia or cnx mutants (Table III). In this screening, some plantlets classified as 'lethal' were rescued. These plantlets, unable to grow on nitrate and to resume normal growth upon transfer to the ammoniumcontaining medium, were submitted to a step of phytohormone-mediated regeneration of shoots, then rooted, and transferred to the greenhouse for progeny testing. O f 17 plantlets rescued from the regeneration process none was found to carry a mutation affecting nitrate utilization efficiency. This step of morphogenesis therefore probably allowed the rescue of plantlets unable to resume
113 Table !II. List of the different mutants obtained in the three selection procedures. Mutants have been referred by the batch number from which they were identified, and by the letter R when selected for chlorate resistance, N for inability to grow on 10 mM nitrate, or a second number when selected for inability to grow on I mM nitrate. Mutants 205R, 205N and 205-1 have been, for instance, isolated from the same M2 population, and statistically should correspond to independent isolats derived from the same M I mutational event. However, when a mutant of one class has been identified in a batch, the probability to isolate a second independent mutant in the same batch is P = 0.12 and this event could have occurred in batch 232 (232N is cnx and 232-3 is nia). The genetic and biochemical data supporting the classification into complementation groups is not presented since it is done essentially as described previously [2]. Chlorate (30 mM)
Nitrate (10 mM)
20 R (nia)
Nitrate (I mM) 24-13 (cnx)
34 N (cnx) 39 R (cnx} 40 R (?) 41 R (cnx A) 56 R (cnx) 60 R (?)
52 N (cnx) 56 N (cnx) 60-5 (nia)
types. The characterization of these mutants, which appear not to be leaky nia or cnx mutants is under way. The frequency of selection of nitrate deficient independent clones is respectively of 45.9/10 000, 52.9/10000 and 54.4/10000 in the three selection procedures. This frequency is probably underestimated since theoretically all the nia and cnx non-leaky mutants identified should have been detected systematically in, at least, the two first rounds of selection as performed on all batches of seeds. The fact that it is not the case results from the limited number of the M2 seeds tested in each batch. These results can be compared to the frequencies of nitrate deficient mutants obtained from M2 screening of barley: 13,1/10 000, pea: 3,5/10 000 [5] and Arabidopsis: 13,3/10 000 [3]. Considering only the nia locus, the frequencies are respectively of 10.6/10 000,14.1/10 000 and 18.1/10 000. The apparently higher mutant frequency observed with N. plumbaginifolia might result from the more efficient rescue of mutant plants under our experimental conditions.
61 N (cnx) 62 R (cnx A) 65 R (cnx A)
Comparison o f cell and M 2 seedling screening 68 N (nia)
70 R (nia) 93 R (cnx)
93 N (cnx) 100-1 (?)
106 R (cnx A) 109 R (?)
205 R (cnxl
105 106 109 110 200
N N N N N
(nia) (cnx) (nia) (nia) (?)
205 N (cnx)
229 N (cnx) 230 (cnx) 232 N (cnx)
203-6 (?) 205-1 (cnx) 209-18 (cnx) 218-9 (cnx)
232-3 (nia) 235-3 (nia)
growth on nitrate for physiological rather than genetic reasons. Two clones, 203-6 and 100-1, were unable to grow at low nitrate concentration, but were able to grow on high nitrate, therefore suggesting that a screening on low nitrate concentration could lead to the identification of new pheno-
Two main conclusions can be deduced from the comparison of the types of mutant isolated either at the cell level or by M2 seedling screening. First the proportion of nia and cnx mutants isolated in both experiments (33% cnx mutants in cell screening, as opposed to 66% cnx mutants in M2 screening) suggests that there is a negative bias in the isolation of cnx mutants from cell screening. This bias is most probably due to the low regeneration efficiency of these mutants. Assuming an equal efficiency of the mutagenesis at the nia locus and at one of the 6 cnx loci, cnx mutants should represent 86% of the population of the nitrate reductasedeficient mutants. The larger proportion of nia mutants always observed, including M2 seed screening, suggests that this locus is more mutable than the cnx loci. Secondly, whatever experimental approach is used for screening, only mutants defective for one or the other of the two main structural components of nitrate reductase have been selected. As opposed to genetic analysis of nitrate assimilation in fungi, where mutations affecting regulatory loci which totally prevent
114 n i t r a t e a s s i m i l a t i o n h a v e b e e n i d e n t i f i e d , s u c h loci h a v e n o t b e e n i d e n t i f i e d i n N. p l u m b a g i n i f o l i a . This can be tentatively explained by three alternative hypotheses: (1) mutations affecting r e g u l a t o r y loci a r e l e t h a l , (2) r e g u l a t o r y g e n e s o r f u n c t i o n s a r e r e d u n d a n t in h i g h e r p l a n t s a n d (3) m u t a t i o n s a f f e c t i n g r e g u l a t o r y loci d o n o t t o t a l l y impair nitrate assimilation. The further characterization of mutants unable to grow on low nitrate concentration might help to test the third hypothesis. Such selective procedures on M2 progeny of mutagenized seedlings should be a method of choice to select nitrate reductase-deficient mutants f r o m p l a n t s p e c i e s r e c a l c i t r a n t t o cell m a n i p u l a t i o n in v i t r o a n d s u b s e q u e n t legumes and monocots.
regeneration,
such
4
5
6
7
8
as 9
Acknowledgements W e t h a n k J. G o u j a u d , Leydecker for technical periments.
A. Douard and M.T. h e l p d u r i n g t h e ex-
10
References 11 I
2
3
A.J. Mfiller and R. Grafe, Isolation and characterization of cell lines of Nicotiana tabacum lacking nitrate reductase. Mol. Gen. Genet, 161 (1978) 67--76. J. Gabard, A. Marion-Poll, I. Ch6rel, C. Meyer, A.J. MiJller and M. Caboche, Isolation and characterization of Nicotiana plumbagin(/olia nitrate reductase-deficient mutants: genetic and biochemical analysis of the nia complementation group. Mol. Gen. Genet., 209 (1987) 596--606. F.J. Oostindier-Brakksma and W.J. Feenstra, Isolation and characterization of chlorate-resistant mutants of Arabidopsis thaliana. Mutat. Res., 19 (1973) 175--185.
12
13
F.J. Braaksma and W.J. Feenstra, Isolation and characterization of nitrate reductase-deficient mutants of Arabidopsis thaliana. Theor. Appl. Genet., 64 (1982) 83--90. A, Kleinhofs, R.L. Warner, F.G. Muehlbauer and R.A. Nilan, Induction and selection of specific gene mutations in Hordeum and Pisum. Mutat. Res., 51 (1978) 29--35. R, Grafe, A. Marion-Poll and M. Caboche, Improved in vitro selection of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia. Theor. Appl. Genet., 73 (1986) 299--304. L. Marton, V. Sidorov, G. Biasini and P. Maliga, Complementation in somatic hybrids indicates four types of nitrate reductase-deficient lines in Nicotiana plumbaginifolia. Mol. Gen. Genet., 187 (1982) 1--3. L.T. Xuan, R. Grafe and A.J. Mfiller, Complemcntation of nitrate reductase-deficient mutants in somatic hybrids between Nicotiana species, Protoplasts, Potrikus, Harms, Hinnen, King and Shilitto, Birkhauser, Basle, 1983, pp. 75. R. Dirk, I. Negrutiu, V. Sidorov and M. Jacobs, Complementation analysis by somatic hybridization and genetic crosses of nitrate-deficient mutants of Nicotiana plumbaginifolia. Mol. Gen. Genet., 201 (1985) 339--343. J. Gabard, F. Pelsy, A. Marion-Poll, M. Cabochc, I. Saalbach, R. Grale and A.J. Mfiller, Genetic analysis of nitrate deficient mutants of Nicotiana plumhagin(fidia: evidei~ce for six complementation groups among 70 classified molybdenum cofactor deficient mutants. Mol. Gcn. Genet., 213 (1988) 2 0 ~ 2 1 3 . A. Strauss, F. Bucher and P. King, Isolation of biochemical mutants using haploid mesophyll protoplasts of tt)'oscTamus muticus. Planta, 153 ( 1981 ) 75 -80. Y. Coic and C. Lesaint, Comment assurer une bonne nutrition en eau et ions mineraux en horticulture. Hortic. Fr., 8 (1971) I1 14. R.R. Mendel and A.J. MiJller, A common genetic determinant of xanthine dehydrogenase and nitrate reductase in Nicotianu tabacum laking nitrate reductasc. Biochem. Physiol. Pflanzen., 170 (1976) 538 541.
109
M2 seed screening for nitrate reductase deficiency in Nicotiana
plumbaginifolia F r 6 d 6 r i q u e Pelsy, J o c e l y n e K r o n e n b e r g e r , J e a n - M a r i e Pollien a n d M i c h e l C a b o c h e Laboratoire de Biologie Cellulaire, INRA-Versailles 78026, Versailles Cedex (France)
Received December 10th, 1990; revision received February 1st, 1991; accepted February Ist, 1991) Nitrate reductase-deficient mutants can be isolated from protoplast-derivcd cells on thc basis of chlorate resistance. Since nitrate assimilation is a highly regulated and tissue specific process, it was expected that mutant selection at the phmt level could lead to the identification of new genes involved in the control of nitrate assimilation. Three selective procedures were compared for the selection of mutants impaired in nitrate assimilation, from M2 seeds: chlorate selection, and rescue of plants specifically unable to grow on low, or high, nitrate concentrations. The rescue of some of the plantlets unable to grow on nitratc was z,chicved through a shoot culture step by treatment with growth regulators. All three procedures led to comparablc frcqucncics of around 5 x 10-3 in the isolation of mutants totally impaired for nitrate assimilation. The majority of these mutants were of the cnx typc, as opposed to mutants selected for chlorate resistance at the cell level, which are predominantly of the tlio typc. ,ia mutants and mutants with leaky phenotypes were also identified, but unexpectedly no other class of mutants totally impaired in nitrate assimilation was identified among isolated mutants. The implications of these data are that M2 seed screening can be used as an alternative route to mutant selection at the cell level for the isolation of mutants totally impaired in nitrate assimilation. This approach can be cnvisagcd for the isolation of nitrate reductase-deficient mutants from species recalcitrant to in vitro manipulation at the cell level and subsequent regeneration into fertile plants. Key words." mutant; selection; chlorate; nitrate assimilation; Nicotiana plumbaginifolia
Introduction
The generation, screening and analysis of mutants is a powerful tool used extensively in the analysis of plant functions. Nitrate reductase-deficient mutants (NR-) have been selected to study the first step of the nitrate assimilation pathway in higher plants. Mutants can be selected from mutagenized cultures of protoplast-derived cells on the basis of chlorateresistance and regenerated into plants. N R mutants have been selected from cell cultures of Nicotiana tabacum and N. plumbaginifolia [1,2] for resistance tO chlorate. These mutants are fully deficient for nitrate assimilation. Alternatively, mutants can be obtained by screening second generation mutagen-treated (M2) seedlings for chlorate Correspondence to: Fr6d6rique Pelsy, Laboratoire de Biologic Cellulaire, INRA-Versailles, 78026 Versailles, France.
r e s i s t a n c e , o r f o r loss o f t h e e n z y m e a c t i v i t y . F o r instance, Arabidopsis, barley and pea chlorateresistant mutants have been isolated from M2 seedlings [3--5]. NADH or NADPH-dependant nitrate reductase-deficient mutant plants of barley and pea have been identified by in vivo NR activity a s s a y o n l e a f f r a g m e n t s [5]. T h e s e p r o c e d u r e s directly lead to the isolation of mutant plants w h i c h a r e u s u a l l y fertile, a n d o v e r c o m e t h e p r o b l e m s w h i c h s o m e t i m e s e x i s t in t h e r e g e n e r a t i o n o f plants from mutant cell lines. S i n c e n i t r a t e a s s i m i l a t i o n is a h i g h l y r e g u l a t e d a n d t i s s u e s p e c i f i c p r o c e s s e d , it w a s e x p e c t e d t h a t m u t a n t s e l e c t i o n a t t h e p l a n t level c o u l d l e a d t o t h e i s o l a tion of new classes of mutants. A drawback of the a p p r o a c h is t h a t m o s t o f t h e m u t a n t s i s o l a t e d b y these screening procedures are leaky, and t h e r e f o r e still a b l e t o g r o w o n n i t r a t e a s sole nitrogen source. This makes difficult the task of their biochemical analysis.
0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
110
Nitrate reductase-deficient mutants are usually classified on two criteria. A first classification is based on the ability of the mutant to retain a xanthine dehydrogenase ( X D H test) activity, an enzyme which shares with nitrate reductase the molybdenum containing cofactor (MoCo). Mutants showing X D H activity, are classified as apoenzyme-deficient mutants (nia mutants). In contrast, those lacking both N R and D H activities are classified as cofactor-deficient (cnx mutants), and must be defective in the biosynthesis or the processing of the MoCo. This biochemical characterization needs to be confirmed by somatic hybridization experiments [l,fr--8] and by genetic analysis to classify the mutants into separate complementation groups [9]. In our laboratory, a collection of 211 chlorate-resistant mutants of N. plumbaginifolia has been selected from protoplast cultures and characterized [2]. All of these were completely defective for the N R activity and carried a monogenic recessive mutation affecting structural genes. By the X D H test, 70 mutants were classed as cnx, which represent 33% of the collection. All fertile mutants were confirmed by genetic analysis and assigned to 7 complementation groups [2,10]: one nia group and 6 different cnx groups (cnx A to F). This paper describes the selection and characterization of nitrate reductase-deficient mutants isolated from M2 progeny of ethylmethane sulfonate (EMS) mutagenized seedlings and selected either for a deficiency in nitrate utilization or for their resistance to 30 mM chlorate. That auxotrophy for nitrate utilization can be used as a selective screen not only at the cell level, as previously shown for Hyoscyamus muticus [11], but also at the plant level is also demonstrated. This latter strategy has been improved with the aim of selecting new classes of mutants affected in the nitrate assimilation pathway. Materials and Methods
Mutagenic treatments Seeds of the haplo-diploidized N. plumbaginifolia cv. Viviani line P B H I were used throughout these experiments. The seeds were mutagenized by soaking in distilled water containing 0.4, 0.5 or
0.6% EMS for 16 h, then washed three times with distilled water, dried, and sown in the greenhouse. Developing plants were then potted individually and grown to maturity. M2 seeds were harvested in bulk from 24 mutagenized plants and surface sterilized.
Mutant selection Approximately 200~300 seeds from each of 118 batches were sown on modified medium B [2] containing 10 m M KNO3 as the sole nitrogen source. In parallel, chlorate selections were carried out on the same batches of seeds, under sterile conditions, on modified medium B containing 10 m M diammonium succinate as the sole nitrogen source, plus 30 m M potassium chlorate. Germinations were carried out in a controlled culture r o o m [10]. After germination on nitrate, NR-deficient seedlings turned white and stopped growing at the cotyledon stage of development. Plantlets displaying this phenotype were rescued on a medium containing diammonium succinate (10 m M ) and KC1 (10 mM) and vegetatively multiplied. The putative chlorate-resistant clones were also subcultured on this ammonium-containing medium. Under selective conditions involving 10 m M nitrate as a selective screen, albino, pale-green, and lethal mutants were discarded on the basis of a similar phenotype on a nitrate- or on an ammonium-containing medium. A third series of selections was performed on 69 of the batches previously used for nitrate and chlorate selection. These selections were carried out on medium B containing 1 m M KNO3. After germination, non-growing plantlets were again rescued on the ammonium-containing medium. Under these selective conditions, plantlets unable to grow upon transfer to ammonium, could occasionally be rescued on the same medium with 0.5 #M naphthaleneacetic acid and 1 #M benzyladenine added. This treatment induced shoot formation, and the shoots obtained were rooted on a hormone-free medium containing a m m o n i u m as the nitrogen source. Plants were further confirmed to be genetically impaired in nitrate metabolism by progeny analysis. Since N R deficient mutants grow poorly on their own roots, putative mutants were routinely grafted onto N. tabacum cv.
111
Wisconsin-38 plants and grown in the greenhouse with a standard nutrient solution containing nitrate and ammonium [12]. Crosses were performed between flowering grafts and reference nia E23 or cnx mutant plants. Detection of X D H activity Young green leaves were collected either on plantlets grown in plastic pots or on grafted rosettes. X D H activity was detected in crude enzyme extract according to the method of Mendel and MUller [13] after electrophoretic separation in non-denaturing polyacrylamide gels and staining in situ for X D H activity. Results and Discussion
Optimization o f mutagenesis Since EMS mutagenesis experiments were not previously described on seeds of N. plumbaginifolia, a true diploid species, a dose-response study has been performed to establish optimal conditions. Three concentrations of EMS were compared with regard to their induction of albino sectors in M 1 plants, and to their remaining fertility. Table I shows that a concentration of 0.5% EMS led to the highest induction of mutated sectors, without much impairment of fertility (97%). Above this concentration, a rapid increase in the proportion of sterile plants was detected (20% at an EMS concentration of 0.6%). We assume that this increase is related to the generation of chromosome breaks and deletions, which are known to strongly reduce the production o f viable pollen. The optimal concentration for EMS (40
Table !. Chlorophyll deficiencies and sterility induced by three EMS treatments on seeds of N. plumbagin(]'olia Mutagenic treatment
Albino chimera (%)
Sterile plants (%)
None 0.4% EMS (32.2 mM) 0.5% EMS (40.25 m M ) 0.6% EMS (48.3 m M )
0.9 2.11 2.26
-0.01 0.03 0.20
mM) is comparable to the routinely used for mutagenesis of an other diploid species, Arabidopsis thaliana [4]. Selection and characterization of mutants resistant to chlorate As the selection for chlorate resistance usually leads to the recovery of mutants totally deficient in nitrate reductase, these mutants are counterselected, in greenhouse conditions, due to their requirement for an organic acid to utilize ammonium efficiently and to grow [2]. To be able to analyse all chlorate resistant plantlets, selections were therefore performed under aseptic conditions, on a medium allowing comparable growth of wild type and mutant plants on ammonium as the sole nitrogen source [2]. Twenty putative chlorate-resistant plantlets were isolated in independent pools of M2 seeds and the progeny of seventeen clones were further characterized (Table II). Among them ten mutants were confirmed to carry a nia or a cnx mutation conferring a total deficiency for nitrate assimilation (Table III). Three clones displaying low fertility were erroneously selected, as their progeny displayed a wild type phenotype. Three clones remain to be characterized. This experiment demonstrated that chlorate can be used to efficiently select mutants totally impaired in nitrate assimilation, from M2 seedlings, since more than 50% of the selected mutated lines were of this type. Selection and characterization of mutants unable to grow on 10 m M nitrate A selection based on the rescue of M2 plantlets unable to grow on nitrate was performed with the aim of increasing the number of loci accessible to genetic analysis. Since N. plumbaginifolia plantlets can be grown on nitrite as the sole nitrogen source, it was expected that, for instance, mutations impairing nitrite reductase activity, or nitrite transport into the chloroplast, could be identified by this approach. Among 53 lines initially selected for their inability to grow on nitrate, 17 turned out to be indistinguishable from the wild type (Table II). It was therefore assumed that their initially impaired growth on nitrate resulted of an independent physiological defect, possibly related to the
112 Table II. A summary of the three selection procedures for obtaining mutants deficient in nitrate assimilation. NS, not scored; NT, not tested. Selective conditions
Chlorate (30 raM)
Nitrate (10 raM)
Nitrate (1 mM)
No. of mutagenized plants tested No. of putative mutants selected Shoot cultures displaying the same phenotype on nitrate or ammonium: Albino, chlorotic or lethals Comparable to wild type Clones rescued on ammonium Clones rescued by regeneration of shoots on ammonium plus hormones Clones transferred to the greenhouse and progeny tested Progeny unable to grow on: Nitrate 10 mM Nitrate I mM Progeny segregationg lethal, albino or pale-green mutants Progeny with wild type phenotype Frequency of mutants defective in nitrate assimilation
2832 20
2832 53
1653 103
NT 17 36 NT
55 12 36 17 out of 55
-18 17
31
50
13 NT I
15 NT 2
7 9 7
3 45.9/10 000
14 52.9/10 000
improper maturation of these seeds during ripening. Two plants expressed a pale-green phenotype which was transmitted to their progeny. As this phenotype was expressed on nitrate as well as ammonium-containing medium, it was not therefore the consequence of a specific defect in nitrate assimilation. The remaining mutants all carried recessive mutations preventing growth on 10 mM nitrate. Ten of them have been classified as cnx on the basis of X D H deficiency, and four as nia by genetic crosses with nia E23. Mutant 200 N remains to be characterized. No mutant defective for nitrite reductase activity has been selected in this experiment. Selection and characterization o f mutants unable to grow on 1 m M nitrate
An other round of selection to isolate M2 plantlets unable to grow on nitrate was carried out with two modifications, on some of the batches of seeds previously used for chlorate and nitrate selection. Lower concentrations of 0.2 or 1 mM nitrate have been used: the lowest concentration
33 54.4/10 000
resulted in the rapid depletion of the nitrogen source from the culture medium, so that it was difficult to distinguish between nitrate depletion and nitrate utilization deficiency by eye screening. This screening was therefore routinely performed on 1 mM nitrate. This procedure again led to the identification of three mutants of the nia type and four mutants of the cnx type, some of these mutants being identified as expected in batches of M2 seeds known from previous selections to contain nia or cnx mutants (Table III). In this screening, some plantlets classified as 'lethal' were rescued. These plantlets, unable to grow on nitrate and to resume normal growth upon transfer to the ammoniumcontaining medium, were submitted to a step of phytohormone-mediated regeneration of shoots, then rooted, and transferred to the greenhouse for progeny testing. O f 17 plantlets rescued from the regeneration process none was found to carry a mutation affecting nitrate utilization efficiency. This step of morphogenesis therefore probably allowed the rescue of plantlets unable to resume
113 Table !II. List of the different mutants obtained in the three selection procedures. Mutants have been referred by the batch number from which they were identified, and by the letter R when selected for chlorate resistance, N for inability to grow on 10 mM nitrate, or a second number when selected for inability to grow on I mM nitrate. Mutants 205R, 205N and 205-1 have been, for instance, isolated from the same M2 population, and statistically should correspond to independent isolats derived from the same M I mutational event. However, when a mutant of one class has been identified in a batch, the probability to isolate a second independent mutant in the same batch is P = 0.12 and this event could have occurred in batch 232 (232N is cnx and 232-3 is nia). The genetic and biochemical data supporting the classification into complementation groups is not presented since it is done essentially as described previously [2]. Chlorate (30 mM)
Nitrate (10 mM)
20 R (nia)
Nitrate (I mM) 24-13 (cnx)
34 N (cnx) 39 R (cnx} 40 R (?) 41 R (cnx A) 56 R (cnx) 60 R (?)
52 N (cnx) 56 N (cnx) 60-5 (nia)
types. The characterization of these mutants, which appear not to be leaky nia or cnx mutants is under way. The frequency of selection of nitrate deficient independent clones is respectively of 45.9/10 000, 52.9/10000 and 54.4/10000 in the three selection procedures. This frequency is probably underestimated since theoretically all the nia and cnx non-leaky mutants identified should have been detected systematically in, at least, the two first rounds of selection as performed on all batches of seeds. The fact that it is not the case results from the limited number of the M2 seeds tested in each batch. These results can be compared to the frequencies of nitrate deficient mutants obtained from M2 screening of barley: 13,1/10 000, pea: 3,5/10 000 [5] and Arabidopsis: 13,3/10 000 [3]. Considering only the nia locus, the frequencies are respectively of 10.6/10 000,14.1/10 000 and 18.1/10 000. The apparently higher mutant frequency observed with N. plumbaginifolia might result from the more efficient rescue of mutant plants under our experimental conditions.
61 N (cnx) 62 R (cnx A) 65 R (cnx A)
Comparison o f cell and M 2 seedling screening 68 N (nia)
70 R (nia) 93 R (cnx)
93 N (cnx) 100-1 (?)
106 R (cnx A) 109 R (?)
205 R (cnxl
105 106 109 110 200
N N N N N
(nia) (cnx) (nia) (nia) (?)
205 N (cnx)
229 N (cnx) 230 (cnx) 232 N (cnx)
203-6 (?) 205-1 (cnx) 209-18 (cnx) 218-9 (cnx)
232-3 (nia) 235-3 (nia)
growth on nitrate for physiological rather than genetic reasons. Two clones, 203-6 and 100-1, were unable to grow at low nitrate concentration, but were able to grow on high nitrate, therefore suggesting that a screening on low nitrate concentration could lead to the identification of new pheno-
Two main conclusions can be deduced from the comparison of the types of mutant isolated either at the cell level or by M2 seedling screening. First the proportion of nia and cnx mutants isolated in both experiments (33% cnx mutants in cell screening, as opposed to 66% cnx mutants in M2 screening) suggests that there is a negative bias in the isolation of cnx mutants from cell screening. This bias is most probably due to the low regeneration efficiency of these mutants. Assuming an equal efficiency of the mutagenesis at the nia locus and at one of the 6 cnx loci, cnx mutants should represent 86% of the population of the nitrate reductasedeficient mutants. The larger proportion of nia mutants always observed, including M2 seed screening, suggests that this locus is more mutable than the cnx loci. Secondly, whatever experimental approach is used for screening, only mutants defective for one or the other of the two main structural components of nitrate reductase have been selected. As opposed to genetic analysis of nitrate assimilation in fungi, where mutations affecting regulatory loci which totally prevent
114 n i t r a t e a s s i m i l a t i o n h a v e b e e n i d e n t i f i e d , s u c h loci h a v e n o t b e e n i d e n t i f i e d i n N. p l u m b a g i n i f o l i a . This can be tentatively explained by three alternative hypotheses: (1) mutations affecting r e g u l a t o r y loci a r e l e t h a l , (2) r e g u l a t o r y g e n e s o r f u n c t i o n s a r e r e d u n d a n t in h i g h e r p l a n t s a n d (3) m u t a t i o n s a f f e c t i n g r e g u l a t o r y loci d o n o t t o t a l l y impair nitrate assimilation. The further characterization of mutants unable to grow on low nitrate concentration might help to test the third hypothesis. Such selective procedures on M2 progeny of mutagenized seedlings should be a method of choice to select nitrate reductase-deficient mutants f r o m p l a n t s p e c i e s r e c a l c i t r a n t t o cell m a n i p u l a t i o n in v i t r o a n d s u b s e q u e n t legumes and monocots.
regeneration,
such
4
5
6
7
8
as 9
Acknowledgements W e t h a n k J. G o u j a u d , Leydecker for technical periments.
A. Douard and M.T. h e l p d u r i n g t h e ex-
10
References 11 I
2
3
A.J. Mfiller and R. Grafe, Isolation and characterization of cell lines of Nicotiana tabacum lacking nitrate reductase. Mol. Gen. Genet, 161 (1978) 67--76. J. Gabard, A. Marion-Poll, I. Ch6rel, C. Meyer, A.J. MiJller and M. Caboche, Isolation and characterization of Nicotiana plumbagin(/olia nitrate reductase-deficient mutants: genetic and biochemical analysis of the nia complementation group. Mol. Gen. Genet., 209 (1987) 596--606. F.J. Oostindier-Brakksma and W.J. Feenstra, Isolation and characterization of chlorate-resistant mutants of Arabidopsis thaliana. Mutat. Res., 19 (1973) 175--185.
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