Theoretical and Applied Genetics 45, 7--20 (1974) ~ b y Springer-Verlag 1974

Quantitative and Qualitative Investigations on the Seed Proteins of Mutants and Recombinants of Pisum sativum* W. GOTTSCHALK and H. P. M(3LLER I n s t i t u t e of Genetics, U n i v e r s i t y of B o n n (GFR)

Summary. The proteins of the seed flour of 26 X-ray induced m u t a n t s and 5 recombinants of the species P i s u m sailr u m were analysed quantitatively and qualitatively. The values obtained were related to the seed yield of the genotypes In this way, the protein yield as well as the production of specific amino acids per genotype were determined. The following results were obtained. 1. The mean values for the character " t o t a l protein content of the seed flour" of the genotypes studied varied between 14 and 23~o. The fasciated m u t a n t 489C produced t8--23~o more seed proteins than the initial line in three subsequent generations. The mean values of some other m u t a n t s and recombinants were 10--17 ~o higher, the lowest value being 27% lower than t h a t of the control values. In the material investigated there is no correIation between seed size and seed protein content. 2. The buffer-soluble seed proteins of 55 m u t a n t s were electrophoretically subdivided in different subfractions and the protein patterns were determined. They are extraordinarily variable. Distinct groups of mutants do not only differ from the initial line but also from one another with regard to the number, position and b r e a d t h of their bands. A correlation between the degree of morphological deviations of specific genotypes and the composition of their seed proteins was not observed. Moreover, differences in the concentration of specific protein fractions between the genotypes were densitometrically ascertained. 3. The globulins and albumins of some genotypes were quantitatively determined and electrophoretieally subdivided into subfraetions which differ between different mutants with regard to their number as well as their concentration. This is especially valid for the albumins which could be essentially stronger subdivided t h a n the globulins. 4. The amino acid spectra of all the m u t a n t s investigated agree qualitatively with the spectrum of the initial line, however, clear quantitative differences in distinct amino acids were observed. The proportion of the essential amino acids is increased in two m u t a n t s b y 5 and 20o/0 in relation to the control line. 5. The protein production of the fasciated m u t a n t 489C was 20-- 70% higher than the corresponding values of the initial line in three subsequent generations regarding its high seed production. The protein yield of the early flowering m u t a n t 46C was about 20~o lower and t h a t of the bifurcated m u t a n t 1201A t 2--31 ~/o higher as related to the initial line. Mutant 1201A shows an equally favourable situation with regard to the t o t a l content of the essential amino acids. Its lysine production exceeded the control values b y 20--40%. 6. The different components of the protein synthesis can negatively be influenced b y the co-operation of the m u t a n t genes. The protein production per plant, the total production of essential amino acids as well as the lysine production are lower in the recombinants 68C/~ 76A and 68C/1201A as compared to the parental nmtants. The combination of genes 68C and 46C does not show a n y neg&tive interactions.

Introduction In modern plant breeding, biochemical methods are i n c r e a s i n g l y b e i n g u t i l i z e d for d e t e r m i n i n g t h e b r e e d i n g v a l u e of species, v a r i e t i e s and s t r a i n s . Besides c a r b o h y d r a t e s , fats, oils, p h a r m a c o l o g i c a l ing r e d i e n t s a n d m i n o r c o m p o n e n t s p r e s e n t in t h e seeds, p r o t e i n s r e p r e s e n t an i m p o r t a n t c o n s t i t u e n t as r e g a r d s t h e i r use in a n i m a l a n d h u m a n n u t r i t i o n (Altschul t965, W o r l d H e a l t h O r g a n i s a t i o n 1965). O n l y r e l a t i v e l y few d e t a i l s are k n o w n so far on t h e p r o p e r t i e s of " p r o t e i n m u t a n t s " a n d t h e i r u t i l i z a t i o n in m u t a t i o n b r e e d i n g . T h e earliest i n v e s t i g a t i o n s h a v e been c a r r i e d o u t in m a i z e (Nelson et al. ~ 965, * The investigations were supported b y the Ministry of Education and Scientific Research of the Federal Republic of Germany and b y the European Atomic Community at Brussels.

Nelson a n d Mertz 1973, M e r t z et al. 1964, 1965). F u r t h e r r e s u l t s in this field o b t a i n e d in d i f f e r e n t crops are s u m m a r i z e d in t h e p r o c e e d i n g s of t h r e e symposia organized by the International Atomic E n e r g y A g e n c y (1969, 1970, t973). M e t h o d i c a l l y it is n o t y e t possible to select p r o t e i n m u t a n t s d i r e c t l y . T h e o n l y w a y to utilize g e n o t y p e s of t h i s k i n d consists in a n a l y s i n g the m u t a n t s a l r e a d y a v a i l a b l e bioc h e m i c a l l y in o r d e r to o b t a i n d a t a on t h e q u a n t i t a t i v e a n d q u a l i t a t i v e s i t u a t i o n of t h e i r seed p r o t e i n s . T h i s h a s been done in Zea (Moss6 et al. 1966, a, b), Oryza ( T a n a k a 1969), H o r d e u m a n d T r i t i c u m ( F a v r e t et al. 1969, G n s t a f s s o n 1969) a m o n g others. B u t also m u t a n t s h a v i n g an u n f a v o u r a b l e p r o t e i n m a k e - u p are of i n t e r e s t b e c a u s e t h e y can be u s e d for s t u d y i n g tile genetic basis of p r o t e i n p r o d u c t i o n a n d p r o t e i n comp o s i t i o n ( B o u l t e r et al. t967, H y n e s 1968, A d r i a a n s e

g

\V.

Gottschalk and H. 1~. Miiller: Seed Proteins of Mutants and l{ecombinants of P i s u m sali,'um

et al. t969, G o t t s c h a l k a n d Mtiller t970, Mfiller 1973,

Mtiller a n d G o t t s c h a l k 1973). Moreover, it is possible to s t u d y the co-operation of m u t a n t genes in this c o n t e x t by c o m p a r i n g the biochemical s i t u a t i o n of r e c o m b i n a n t s a n d thei p a r e n t a l m u t a n t s . I n the present p a p e r an a c c o u n t of the relations between seed p r o t e i n c o n t e n t a n d seed yield of a group of m u t a n t s a n d r e c o m b i n a n t s of P i s ~ m , sativ~m~ were a n a l y s e d q u a n t i t a t i v e l y as well as q u a l i t a t i v e l y . Also, an a m i n o acid analysis of the p r o t e i n s was made in order to s t u d y the action of the m u t a t e d genes on the composition of the seed proteins.

Materials and Methods

%

200 190

180 170 160 150 140 130

110 100

For the genetic and biochemical investigations 20 Xray-induced m u t a n t s and 6 recombinants of the variety 'l)ippes gelbe Victoria' of P i s u m salivum were used. The following genotypes are of particular interest because of their useful breeding characters: m u t a n t 46C: early flowering and ripening; mutant68C: increased number of ovules per ovary ; m u t a n t 176 A: small leaflets, stipules, flowers, pods and seeds; mutant251A: apical part of the stem relaxed fasciated, flowers and pods distributed over a relatively long part of the stem ; n m t a n t 489C: apical part of the stem strongly fasciated, all the flowers and pods clustered at the top of the plant (the two genes for stem fasciation belong to a series of multiple alleles) ; m u t a n t / 000 : large seeds ; m u t a n t 1201A : dichotomous stem bifurcation, reduced penetrance ; recombinant R 1 76X: narrow leaflets, stem fasciation, late flowering (from a spontaneous hybridization of m u t a n t s 176A x x 489C) ; recombinant 1{ 177: stem bifurcation, stabilized penetrance (selected in an F,-generation 489Cx 120~A); recombinant R 300: homozygous for genes 68C and 46C; recombinant R 320: homozygous for genes 68C and 176A ; recombinant R 35O: bomozygous for genes 68C and 1201A;

90

2.

B iochemica! )t,lethods

The total nitrogen content of the seed meal (dry weight basis) was determined by the Kjeldahl method. The protein content was calculated by multiplying the nitrogen value by 6.25 (Keil and Sormova 1965). The soluble proteins were determined by the method described by Lowry et al. (t951).

recombinonts

210

1. The 3/[uta~zts

The seed yields of these genotypes are graphically illustrated in fig. t. The small-grained m u t a n t s 251A and 489C show an extraordinarily high seed production as a consequence of the stem fasciation. The seed production of the recombinants is in general less than that of the parental mutants. Moreover, the yielding capacity of recombinants ]{ 176X and I{ 177 due to an extremely small size of their seeds is negatively influenced.

mutants

- - - -

80

70 60

I

t

Fig. 1. The seed production of some pea mutants and recombinants in subsequent generations, leach point represents the mean for the character "number of seeds per plant" as percentage of the corresponding value of the initial line The albumins and globulins were extracted from the seed meal according to Danielsson (1949, /956). The protein content was determined by the Folin test (Lowry el al. 1951). The analysis of the globulin fractions was carried out on gels containing 7.50/o acrylamide according to Bloemendal (1967) and Maurer (1968). The albumins were analysed using the method of Loeschke and Stegemann (1966). The destained gels were compared, the Rp-values determined and the banding pattern schematically drawn. Furthermore, the gels were scanned densitometrically using the Chromoscan (Joyce-Loebl). The gel patterns of the different genotypes were compared with one another on the basis of optical density curves (Maurer 1968). For comparison, they were evaluated by measuring the area of the peaks (Mtiller 1970). Samples of seed meal were hydrolysed according to Barton-\Vright (t 952), Neff and Sormova (1965) and Horn el al. (1947, 1955). The hydrolysates were analysed in an amino acid analyser (Aminolyzer, Optica) according to the procedures given by Mondino (1967, 1969a, b).

Results The a m o u n t of the seed p r o t e i n s as well as their q u a l i t a t i v e composition is an i m p o r t a n t c o m p o n e n t for the yield of m a n y c u l t i v a t e d p l a n t s . Their analysis can be carried out in two different ways. One w a y is to use equal a m o u n t s of seed flour of different m u t a n t s for d e t e r m i n i n g the p r o t e i n c o n t e n t a n d the p r o p o r t i o n of specific a m i n o acids. The a d v a n t a g e of this m e t h o d lies in its possibility to compare a large n u m b e r of different g e n o t y p e s with one another, b u t the other c o m p o n e n t s of yield c a n n o t be considered in this way. The other possible a p p r o a c h is to combine this m e t h o d with an e s t i m a t i o n of factors such Theorel. A p p l . Ge,netics, Vol. 45, At~ I

\V. Gottschalk and H. P. Miiller: Seed ['roteins of Mutants and Recombinants of Pisr Fig. 2. The frequency distribution of ~he protein content of 1450 genotypes of the world collectionof Pisurn sativztm (below) in comparison to 26 mutants and recombinants of our assortment (above). I~orizontal axis : Total protein content ill percent of the dry weight of the seeds. Vertical axis: Proportion of the genotypes in the different protein classes

salivum

9

voriety "Dippes getbe Viktorio" (26 genotypes)

nl

tip world collection Pisum sotivum (1/.50genotypes)

~N

__o0111

1/. 15 16 17 18 19 20 21 22 23 24 25 26 2? 28 29 30 31 32 33 3/, 35 36 317 38 39

as the number of seeds per plant and tim seed size which take part in seed production of the plant in order to calculate seed weight per plant. This value can be related to the protein and amino acid values. In this way, mean values for the protein production per plant as well as for the total production of distinct amino acids can be obtained. The yielding components mentioned above vary considerably in different mutants in subsequent generations and thus protein production is deviated positively as well as negatively. The present work gives an account of the results got after utilizing both the above given methods for a group of mutants and recombinants of Pisum satiU'lt, N$.

1. The Quantitative and Qualitative Analysis o~ the Proteins o/the Seed Flour a) The Total Seed Protein Content. The legumes because of their high protein content are of great economical importance especially in countries with widespread vegetarian diet. The total protein content of the seeds of most cereals varies between 9 and l'heoret. Appl. Ge~etics, l'ol. 45, No. 1

96 protein

13 precent of the dry weight. In contrast to this, in legumes like Glycine max and Arachis hypogaea the values reach up to 45 percent and are comparable to some protein-rich oil crops~such as Sesamurn, Linum, Gossypium, Helianthus (see Altman and Dittmer 1968). The garden pea occupies an intermediate position in this respect by showing a variation from 1 4 - - 3 9 percent. Majority of the cultivars and strains of the world collection tested so far have a total protein content of 22--29 percent. The distribution of the character "total seed protein content" is graphically presented in the lower part of figure 2. The diagram is based upon the mean values of 1450 different genotypes of the world collection of P i s u m sativum without consideration of mutants (Slinkard 1972). The initial line 'Dippes gelbe Viktoria' of Pisum sativum used for our radiation genetic experiments has a total protein content varying between 19 and O / 22,o and occupies the lower protein range in the world collection. Precise comparison of our mutants and recombinants with the existing cultivars and strains of the world collection is difficult because only

10

\V. Gottschalk and H. P. Miiller : Seed ;Proteins of Mutants and ]~ecombinants of P i s u m sativum

group

I

group I I

120

m))

110 ,

:

m 68C 70 71

. [.

1201A

68/1201

68(;

/.6C

70 71

?0 71

70 71

70 71

Fig, 3. The tatar protein content in the seed flour of Pisum mutants and recombinants of three different hybrid combinations as related to the values of the initial line = 100% in two subsequent generations. Ineach combination the recombinant is compared with its two parental mutants

groupm

68/46 70 71

26 genotypes of our assortment have been analysed so far. If we relate this relatively small number of findings to the corresponding data of world collection (upper part of fig. 2), the balance appears to be negative. However, considering the relatively bad biochemical situation of the initial line a considerable variation of the mean values of the different genotypes is evident. This shows that the amount of seed proteins can considerably be altered in positive or negative direction under the influence of a single gene or a small number of genes. The most protein-rich genotype of our assortment is the fasciated m u t a n t 489C that produces 18--23% more total protein as compared with the initial line related to equal amounts of seed flour. In this value, the high seed production of the m u t a n t is not yet considered (see section 2 of the present paper). Some other mutants and recombinants show likewise an increase of the protein content by I0--17~o. The lowest value of 73.40/o of the control value was found in the recombinant R177 having a dichotomous stem bifurcation in combination with a high penetrance of one of the two genes involved. A comparison of some recombinants with the respective parental mutants is of particular interest with regard to the co-operation of mutated genes as far as the protein synthesis is concerned. The results of three different hybrid combinations are represented in fig. 3. Mutant 68C was one of the parents in all the three crossings; mutants i 2 o I A (stem bifurcation), 46C (earliness) and 176A (small grains) were used as the second partner. The seed protein content of m u t a n t 68C is similar to that of the initial line while m u t a n t 12olA showed varying values in t970 and t971 (fig. 3, left-hand part). The recombinant R 35o homozygous for genes 68C and 1201A shows in both these growing seasons the protein values equal to that of m u t a n t I2o1A. Thus, in 1971 the full protein content of m u t a n t 68C was not reached in the recombinant. From these findings certain negative interactions between the two mutated genes become discernible. As this situation was only realised in one of the two years of study it can be assumed, that the gene interaction is vulnerable to some environmental factors. Further work in this direction is in progress. The same situation is valid for hybrid group I I I (fig. 3, righthand part).

68C

1?6

71 72

71 72

68/176 71 72

Also in this case the high protein content of the smallgrained m u t a n t 176A is not reached by the recombinant R 32o ( = 68C/176A). The protein level of this recombinant is likewise similar to that of the parental m u t a n t which has a low protein content. Group I I contains two mutants of equal protein content. The values of the recombinants lie in the same range; thus, the genes involved do not show any positive or negative interaction with regard to the character "total content of the seed proteins". In cereals a negative correlation between seed size and protein content is known since long; the smaller the seeds, the higher their total protein content. This correlation could not be confirmed in our material. The distribution of the mean values of 23 mutants and recombinants seems to suggest a certain trend in this direction. However, it was not possible to support this assumption statistically by means of the regression analysis separately for mutants, recombinants and the total material. Thus, it can be concluded, that the selection of small-grained pea mutants does not result in a selection of protein-rich genotypes. Also, there are no statistically significant correlations between seed size and protein content. A particularly clear example in this respect is the recombinant R ~77 mentioned above. It is one of the most small-grained genotypes of our assortment % i

F

9

io 9

E

110 er

"~ 100

1o 3

g| 90

o .g

o

o

o

9

o o

i

"6 80 50

60

70

80

90

100

llO

thousand kernel weight (96 of the initial line)

Fig. 4. The relations between thousand grain weight and total protein content of the seeds of 23 mutants and recombinants of Pis*~m sativum Theorel. A p p l . Genetics, l'ol. 45, No. 1

\V. Gottschall{ and H. P. Miillcr : Seed Proteins of Mutants and Recombinants of Pisum sati,,~t,~ character

mutant

banding pattern

I

initial Line 176

SmaLLgrains

1001

Large grains

235

chLorophyLLdefect

46

earliness

94

fLower anomaly

I000

Large groins

157

stem bifurcation

~,5

waxtess

333

chLorophyLLdefect

&l

desynapsis

137

chLorophyLLdefect'

489

fasciation

75 116

Long internodes waxtess

tI

III I I I I

I I I

II I I II I II I I I I I I I I

I IIII I III I I III I I III

I I

II I

II I I I IIIIII II I I I I I III I I I IIIII I IIIII

II I I I IIIII I I I IIII I I I I III I I I III I I I II

I I I I I II

I

I I I I III I I II I I II I I I II I I I

II II I I II IIIII I I

I I I

I I I I I

I III

origin Fig. 5. T h e b a n d i n g p a t t e r n s of t h e soluble seed p r o t e i n s of s o m e pea m u t a n t s in c o m p a r i s o n to t h e initial line

(thousand kernel weight = 75.2% of the initial line), but it shows the lowest protein content of all genotypes studied so far (73.4% of the initial line). On the other hand, the large-grained m u t a n t No. 10o0 exo/ eeeds the control values only by 6/ob) The Protein Patterns. Using the acrylamidegel-electrophoresis it is possible to separate the soluble seed proteins in a series of distinct fractions. After t r e a t m e n t with specific dyes they become discernible in the gel in a specific distance from the starting point. In this way, a protein pattern occurs which is specific for each genotype with a high degree of reproducibility. So far, 55 m u t a n t s of our assortment have been analysed in this respect. As shown in fig. 5, an extraordinarily broad diversity of the protein patterns was obtained. Using this method, the total number of bands varied between 12 and 18 in the m u t a n t s studied. In some strikingly broad bands further separation using a more sophisticated method seems to be possible. Using standardized conditions, 55 different protein patterns were obtained for the 55 different mutants. 14 of them are schematically illustrated in fig. 5. Let us compare m u t a n t 94 and the initial line as an example for the diversity of two genotypes. The m u t a n t has eight bands more; furthermore, it is characterized by a particularly broad band t h a t in the control material exists in the form of a narrow streak Theater. Appl. Ge,~elics, Vat. 45, No. t

(fig. 5, group II). Moreover, rapidly moving fractions occurring in the m u t a n t are absent in the initial line (righthand part of the figure). Thus, it is evident t h a t the seed proteins of m u t a n t 94 are partly composed of certain different subfractions than those of the initial line. A single gene pair is responsible for these differences. A similar situation was observed for m a n y other m u t a n t s analysed earlier (Gottschalk and Mtiller 1970). Interestingly, this is also valid for typical " m i c r o m u t a t i o n s " that can be identified in segregating families only with great difficulties. Also with regard to their seed production they are in m a n y cases comparable with the initial line. Thus, there are no correlations between the degree of the morphological deviations and the composition of the seed proteins. For further details concerning the differences of the protein patterns see fig. 5. The findings just mentioned refer to the qualitative situation of the seed proteins. Furthermore, a quantitative evaluation was carried out by a direct densitometric scanning of the gels. From the intensity of the staining of the various protein fractions certain conclusions concerning the respective quantities can be drawn. Thus, the densitometrically scanned absorption curves can be used for calculating the quantitative situation. In fig. 6 the absorption curves of two m u t a n t s are compared with that of the initial line. Strong differences between the three genotypes are particularly discernible in the lefthand part of the

12

W. Gottschalk and H. P. Mfiller: Seed Proteins o f Mutants and Recombinants of P i s u m satin,urn

- -

initial

.......

mutant

--

mutant

:.

--

curves, i.e. in the pattern of the slowly moving proteins. The curves show, that proteins with similar moving speed can be present in differing amounts depending on the presence of m u t a n t genes. Analogous findings have already been obtained with regard to other mutants of our collection earlier (see Gottschalk and Mtiller 1970).

Line

176

",,'! :..:

:

c) The Protein Fractions. The seed proteins of the Legctmi~osae consist mainly of tim salt-soluble globu9

I i i

J

I Ill I ii/ Nf

..

':

:,

,,....

/rig. 6. The direct densitometric evaluation of the protein bands of the initial line and of mutants 46C and 176A

initial Line and mutants initial Line

I 1201A

I 46

251A

I 176A

I 489C

recombinants R177

1 2 3 4 5 6 7 8

I R65

I

12345678 protein fractions

I R200

12345678

lins and the water-soluble albumins as the main fractions. 75% of the total protein of our initial line are globulins. They represent the most essential reserve proteins of the plant which are mobilized during seed germination and are responsible for the maintainance of the earliest stages of the ontogenetic development. The albumins are the enzymatically active seed proteins mobilizing the globulins. Both the fractions can be eleetrophoretically subdivided into their subfractions. An evaluation of the globulins of some mutants and recombinants has shown that the genotypes differ from one another and from the initial line with regard to the number of subfractions as well as their concentration. Considering the genotypes analysed eight different subfractions could be found according to their position at the gels. Their number and concentration vary in different geL notypes. Some examples are given in fig. 7. The initial line shows 4 bands of distinguishing breadth and migration velocity at the gel. They are given in figure 7 according to their distance from the starting point. The height of the columns is a measure for the concentration of the respective fractions and has been obtained by scanning the gels. The area of the various peaks was planimetrically determined and was related to the total area of a l l the peaks present ( = I oo%). If we compare the fasciated m u t a n t 251A with the initial line there is a concordance of the two genotypes with regard to the number of the globulin subfractions, but fraction no. 4 is present in mutant 251A in an essentially lower concentration. The opposite relation is valid for fraction no. t while fractions no. 6 and 7 differ only slightly in the two genotypes. The recombinant R 177 belongs to the same group. The second group of genotypes (fig. 7, middle part) shows only three

Fig. 7- The electrophoretically obtained globulin subfractions of five mutants and three recombinants of Pis~tm saliwtm in comparison to the initial line. Tile height of the columns were obtained by a densitometric evaluation of the b~mds. Tile sequence of the columns corresponds to the position of the bands in the gels Them'eL. Appl. Ge~elics, Vol. 45, .\:o. 1

W. Gottschalk and H. 17. Miiller: Seed Proteins of Mutants and lr subfractions which nearly agree in m u t a n t s no. 12olA and 176A (subfractions no. 1,4, 6). In the recombinant R 65, however, the subfraction no. 6 is absent, but the subfraction no. 2 is present which could not be found in the other genotypes analysed so far. The early flowering m u t a n t 46C with its subfractions 1,4, 7 belongs likewise to this group. The fasciated n m t a n t 489C having 6 globulin subfractions with a characteristic distribution is of particular interest. Subfraction no. 8 of this m u t a n t could not be found in the other genotypes studied. The comparison of the 5 n m t a n t s and 3 recombinants of figure 7 shows clearly that the globulins in legumes do not consist of only two subfractions as described in the literature (Danielsson 1949, 1956). Furthermore, the figure demonstrates strong genetically conditioned variations of tire globulin patterns. The subfractions of the albumins can be stronger separated from one another using the disc gel electrophoresis. 35 bands were found by means of staining with Coomassie Blue. Also in this respect, clear quantitative differences of specific subfractions were stated between different m u t a n t s of our assortment. Detailed investigations are in progress; the results will be published elsewhere. Tile co-operation of some genes has been tested with regard to the quantitative distribution of the globulins and albumins b y comparing the recombinants with their parental mutants. The data of two hybrid groups available so far show a diverging behaviour (fig. 8). Let us at first consider group I I (crossing 68C x 46C). The globulin content of m u t a n t 68C lies only slightly, the albumin content considerably below the values of the initial line. Mutant 46C possesses higher amounts of both these fractions. The recombinant R 3oo (~-- 68C/46C) corresponds to m u t a n t 46C with regard to the globulin as well as the albumin production. Thus, gene 46C exerts its full action on the globulin and albumin production even when gene 68C is present in the genome. Hence, the negative influence of gene 68C on the two protein fractions is ineffective in the recombinant 68C/46C. The situation is more complicated in the hybrid group I (68C • 12o1A; fig. 8). The globulin production is clearly reduced under the combined action of both the genes; the value for recombinant R 35o ( = 68C/1201A) is only 82% of the corresponding mean of the initial line while the values of the parental m u t a n t s are 93 or 1 0 2 ~ respectively. F r o m these findings an interaction between the two m u t a n t genes influencing the globulin production negatively becomes obvious. The albumins, however, are in a different way influenced by the combined action of the two genes. The albumin content of the recombin a n t R 350 ( = 68C/12o1A) lies considerably below of t h a t of m u t a n t 12olA, but it is higher than t h a t of 68C. Thus, the negative effect of gene 6SC gets reduced by the presence of gene 1201A. Theoret. Appl. Genetics, Vol. 45, No. 1

of Pism~a sati~um

group I

68

1201 68/

gr0upll

globulins

albumins

globulins

68

13

1201 68/ 1201

1201

68

46

68/ 46

otbumins

68

~6

6e/ 46

Fig. 8. The quautities of globt lins and a l b u m i n s of two h y b r i d c o m b i n a t i o n s as related to the control wflues of the initial line --= I 0 0 /o/ o . The values of the r e e o m b i n a n t s are in both the groups c o m p a r e d w i t h those of the parcnta[ m u t a n t s

d) The Amino Acid Composition o~ the Proteins. The proteins of the legumes consist of 20--25 amino acids, t 6 of them can be quantitatively determined by means of an amino acid analyser using acid hydrolysates. As expected, no qualitative differences between different genotypes could be found considering the analytical data of the initial line, four m u t a n t s and some recombinants. All the amino acids are present in their seed proteins, but there are clear genetically conditioned quantitative differences with regard to specific amino acids. The amino acid spectrum of the initial line and of m u t a n t s t201A, 68C, 46C and 176A is graphically illustrated in figures 9 and l o separately for nonessential and essential amino acids. The amino acids are arranged in the same order in which they were registered b y the amino acid analyser. The figure 16 m

15

oc 13 E~ 12 "E

o 9 g.

7

g, s

~3 2 1 ASP

lltllllll "il J lallHmll

[ SER

GLU,

Fig. 9. The percentage of the non-essential amino acids as related to tile total a m o u n t of the a m i n o acids in the initial line and in four 1)isu*~; m u t a n t s . Column ~ : initial line; Column 2: m u t a n t 12ol A, Colunm 3 : m u t a n t 68C; Column 4 : m u t a n t 46C; Columlt 5: m u t a n t 176A

14

W. Gottschalk and H. P. Miiller: Seed Proteins of Mutants and Recombinants of P i s u m s a l i w t m Fig. 10. The percentage of the essential amino acids as related to the total amount of the amino acids in the initial line and in four Pisum mutants (arrangement as in figure 9)

12 11 9g ~0

~7

:I I

o

,Ii

llml

5

IMI I

g 3 O

THR

VAL

ILEU I

LEU

,,

I PHE

shows the quantitative situation of the various con> ponents of the total hydrolysate which is more or less similar in all the genotypes studied. Glutamic acid occupies the highest proportion of the total amount of the amino acids with t 3-- 16 percent while the proportion of methionine is very low reaching a mean of nearly one percent only. With regard to the problem discussed in the present paper the genetically conditioned differences between the various mutants are of particular interest. They are low for most of the amino acids; however, clear deviations could be found in,single cases. Mutants 1201A and 176A, for instance have higher leucine and arginine than that in the other three genotypes but the amounts of glutamic and aspartic acid are reduced in both of these mutants. The small-grained m u t a n t 176A shows considerable deviations with regard to specific amino acids as compared with the other genotypes studied; phenylalanine is present in an increased and proline in a reduced amount. A corresponding quantitative variability was likewise observed for the other nmtants analysed presently (fig. 9 and t 0). The protein quality depends on the proport:ioll o~ the essential amino acids constituting the seed proteins. Therefore, the influence of some m u t a n t genes on the production of this group of substances should be specifically discussed considering again the cooperation of the genes involved. In the upper part of fig. 11 the total content of all the essential amino acids of the three hybrid combinations mentioned in the preceding sections is illustrated. Mutant 68C does not differ in this respect from the initial line. Mutant 46C produced four percent less, mutants 1201A and 176A five and twenty percent more essential amino acids, respectively. All these values refer for equal amounts of seed meal while the seed yield of the respective genotypes is not yet considered. The co-operation of gene 68C with genes 12olA, 46C and 176A leads to different results. The recombinant R 35o ( = 6SC/12olA) shows a similar produc-

tion of essential amino acids as compared to m u t a n t 1201A. Thus, there is no negative interaction between genes 68C and 12olA in this recombinant. The combination of genes 6SC and 46C results in an even more Iavourable situation because the recombinant R 300 ( ~ 68C/46C) exceeds both the parents by 25

]"ig. ~ 1. Comparison of the total a m o u n t of the essential &lllino acids and the lysine and methionine c o n t e n t in three different h y b r i d combinations. All the values are related to the initial line; the r e c o m b i n a n t s arc again c o m p a r e d with the respective parental lnutants

Theoret. d p p l . Genetics, l'ol. 45, No. I

W. Gottschalk and H. P. Miiller: Seed Proteins of Mutants and l(ecombinants of Pisum sativum to 30%. Therefore, its seeds should be valuabJe especially as far as its nutritional status is concerned. In the third hybrid combination (68C x 176A) a compromise is reached between the two genes involved for the total content of essential amino acids lies between corresponding values of both the parental mutants. Finally, the co-operation of the genes just mentioned with regard to two specific amino acids will be described. We have chosen methionine as the limiting essential amino acid of the proteins of the legumes and lysine because of its high nutritive importance. The quantitative situation of both these amino acids in the mutants and recombinants studied is graphically illustrated in the middle and lower part of fig. 11. The simplest situation is realised in the hybrid combination I I (68C • 46C). Both the mutants are producing both these amino acids in essentially higher quantities as compared with the initial line. Thus, they can be considered positive as far as their nutritional value is concerned. The combination of both the genes in the same plant organism has neither positive nor negative consequences. In hybrid combination I (68C • 12o1A), however, the lysine production of the recombinant is similar to that of the low parental m u t a n t (120~A). Thus, the favourable conditions of n m t a n t 68C do not become effective in this combination. The same situation is realised in hybrid combination I I I (68C • 176A). In contrast to the lysine behaviour the methionine production shows a more uniform behaviour. In the recombinants R 350 (68C0 1201A) and R?2o (6SC//76A) that gene dominates which is responsible for the formation of the higher methionine amount. 2. The

Relations

between Seed Production

Yield and

Protein

All the data presented in the preceding sections on proteins, protein fractions and amino acids are related to equal amounts of seed flour irrespective of the seed production of the genotypes studied. Yrom a breeder's standpoint, however, the seed protein content should be related to the seed yield of the genotypes. Using the criteria " n u m b e r of seeds per plant" and "seed size" (thousand grain weight) the mean for the character "seed weight per plant" can be calculated. If we relate this value to the protein content of the seeds the protein production per plant can be estimated. Let us demonstrate the relations between the criteria just mentioned using a clear example. The highest yielding genotype of our assortment is the fasciated m u t a n t 489C. Considering 9 generations the mean values for the charactel "number of seeds per plant" varied between 132 and 212 percent of the corresponding means of the initial line. Fig. 12 gives the mean values for the years 1969--197 I, these vary between 152 and 212% of the control values. The seeds are very small, but the seed yield of the m u t a n t Theater. Appl. Genetics, l'ol. 45, No.

1

is higher than that of the initial line in majority of the years. This is because of the high seed number per plant of the mutant. The seed protein content is 2o to 25~ higher resulting in 2o--70% of increased protein production per plant over that of the initial line. A second fasciated m u t a n t of our collection shows a similarly fawmrable situation (Miilter and Gottschalk 1973). The nutritive value of these seeds tested by Eggum in Denmark through rats feeding teclmique indicated no differences between m u t a n t and initial line. Thus, the increased protein production is in this case reallv equivalent with an increase of biologically utilizable protein. % 21o

]

numberof seeds per plant

200 190

]

weight per 1000 seeds

180

17( 1B(

]

seed weight per plant

J

protein content per lOOg seed flour

]

protein weight per plant

'td 31 2 1 I0 g E 7 E

11|

i

Fig. 12. Comparison of different criteria of the protein yield of nmtant 489C in three subsequent generations Let us now discuss the correlations between seed production and different criteria of the protein production in those mutants and recombinants already described in the preceeding sections. From the protein content of the seed flour and the seed production of the plant the total amount of the seed proteins per plant was calculated. Moreover, the total amount of seven essential amino acids was commonly considered (valine, leucine, isoleucine, phenylalanine, threonine, methionine and lysine); methionine and lysine were separately evaluated. The mean values for all these criteria are graphically illustrated in figure 13 considering three mutants and two recombinants in two subsequent growth seasons. All the means are related to the corresponding values of the initial line. Let us first regard the total protein production of the five genotypes. As expected, the values of the initial line for the characters "number of seeds per plant", "seed size" and "protein content of the seed flour" do not fully resemble in different growth seasons. If we calculate the mean values for the character "protein production per plant" from these traits

16

W. Gottschalk and H. P. Mtiller: 5ccd Proteins of Mutants and l{cconlbinats of Pisu~z satic,um

tile following values were obtained: -

-

-

-

197o: 1.095g, 1971: 1.562g.

The difference is extraordinarily high. In 197t, the plants of the variety 'Dippes gelbe Viktoria' produced about 43 percent more seed proteins than in 197o. The m u t a n t s and recombinants investigated show likewise clear differences in both these years, but in the opposite sense; all the values for 1971 are lower than those for 197o. Mutant 68C has been used for crosses with other beneficial m u t a n t s of our collection because of its genetically conditioned increase of the ovule number per carpel. Tile protein content of its seed flour is similar to that of the initial line (1ol.9 and t o l . o % in 197o and 1971). However, the protein production per plant varied considerably in both the growth seasons as a consequence of strong differences of the seed size (protein production = 131.1 and 92.4~}/o of the initial line in 197o and t971, respectively). A similar situation was observed for the early flowering m u t a n t 46C. The negative factor in this case is the reduced number of seeds per plant (79 and 72% of the control values, respectively). Therefore, its total protein production was far below the control values of the initial line (77 and 76~o in 197o and 1971, respectively). In contrast to the two genotypes just mentioned the situation of the bifurcated m u t a n t 12o1A is more favourable. In 197o, the protein content of its seed meal was about equivalent to that of the initial line, but in 1971, it w a s 12~ lower. However, tile stem bifurcation results in an increase of the seed production per plant. Considering 14 generations, the mean values for the character "seed number per p l a n t " varied between 92 and 121~ of the control values. With regard to the seed size, only minute deviations from the initial line were observed. Therefore, favourable values for the character "seed weight per p l a n t " were obtained exceeding the control b y about 30~ in 1970 and 1971. Considering the thousand grain weight and the protein content of the seed meal the total production of seed proteins per plant was 31 and 12~ higher than t h a t of the initial line in both the generations tested. All the data mentioned refer to the mutants. By combining the m u t a n t genes it was our aim to produce recombinants which could be of interest in pea breeding. The low seed production of the early flowering m u t a n t 46C should have been improved b y an increase of the seed number t h a t is inherited through gene 68C. However, this was not possible because of certain negative interactions between the respective genes (Gottschalk, 1972). Considering five subsequent generations the recombinant R300 ( = 68C/ 46C) reached values between 72 and SS~ of the corresponding values of the initial line only for the cha-

r a t t e r " n u m b e r of seeds per plant". Tile protein content of its seed meal is equivalent to that of the control (1o4.3 and to3.9% , respectively). However, a decrease of the protein production per plant m a y occur as a consequence of varying seed sizes in subsequent years. In t97o, the protein yield of the recombinant was 98.6~o of tlle control value, in 1971, it was only 8t~ Thus, the differences between the recombinant and the initial line are considerably lower if we not only regard the seed but also the protein production. Therefore, the breeding value of the recombinant is improved b y its favourable protein situation. The recombinant R 35o ( = 68C/12oIA) shows a different initial situation which, however, does not result in an inlprovement of the protein production. The characters "increased seed number per pod" and "increased pod number per plant" are combined in the plants of this genotype. There is no negative interaction between the two genes involved; nevertheless, their combination does not result in tile effect expected. With regard to the number of seeds per plant the mean values are similar to those of the initial line considering 5 generations, but the seeds are smaller. In this way, tile seed weight per plant decreases to about 9o% of the corresponding control values. The protein content of the seed flour was 1o2.4~ of the values of the initial line in 197o, but in 1971 only $7.6~ Furthermore, if we consider the reduced seed size, even stronger reduction of the protein yield per plant is reached (92 and 78% of the control values for 197o and i 97 l, respectively). More i m p o r t a n t than the total protein content determined by means of the Kjeldahl method are the essential amino acids because they exclusively are of importance for the protein quality and the nutritive value of the seed proteins. Let us first consider the total amount of the seven essential amino acids mentioned above. The mean values of the initial line were o.5o9 (197o) and o.726g per plant (1971), this is a proportion of 46.5~ of the total seed proteins in both the years. The three m u t a n t s investigated show a very different behaviour in this respect. Mutant 68C reached the control value in 197o; in 1971, however, the total amount of the seven amino acids was only 81 ~/o of the corresponding amount of the initial line. Mutant 46C showed in both the growing seasons equally negative values (69 and 68~u of the control, respectively). In contrast to this, the bifurcated m u t a n t 12olA exceeded the initial line not only in 197o but also in 1971 when all the m u t a n t s and recombinants tested showed considerably lower values than in t 97o. The total amount of the essential amino acids produced from this m u t a n t was 24 (197o) and 7 (1971) percent higher as compared to the initial line. Lysine and methionine have been separately evaluated from the pool of the essential amino acids for the reasons already mentioned. With regard to Theoret. Appl. Genetics, Vol. 45, No. 1

\V. (.;ottschalk and H. 1'. Mtiller: Seed Proteins of Mutants and Recombinants of Pisum sativt~m

pro'.'ocon'.n in 100 gms of

17

I 5..dProt"n I omoonto'I'ota"'o'c~176I to'~

per plant

seed flour

content per

essential amino

plant

acids per plant

per plant

content per plant

1970

1971

a

b

c

d

e

I

a

b

c

d

e

I a

b

c

d

e

l

a

b

c

d

e

l a

b

c

d

e

I

a

b

c

d

e

l;ig. 13. Comparison of different criteria of the protein yield in some pea mutants and recombinants in two subsequent generalions. All ~.hevalues are related to the corresponding values of the initial line loO%. a: mutant 68C ; b : rout;rot 46C ; c : mutaz~t 12(;IA ; d: recombinant R30O (-- 68C/40C) ; e: recombinant 1{.35o(= 68C/12ol :\) upper part: 197o, lower part: 1971 the lysine production n m t a n t 6SC is equivalent with the initial line while both the values of n m t a n t 46C are v e r y low (middle p a r t of fig. ~3). M u t a n t 1201A shows a v e r y favourahle situation in this concern. its lysine production was 4o% 097o) and 2o~ (1971) higher. The methionine production is in all the three m u t a n t s in comparison to the initial line higher t h a n the lysine p r o d u c t i o n ; the m u t a n t s show a similar relation. Mutants 68C and 12olA exceeded the control values considerably. The situation in the two r e c o m b i n a n t s R 3oo ( = 68C/46C) and R 35o (-- 68C/ t201A) is less favourable. The total a m o u n t of the essential amino acids and the lysine production lies below to t h a t of the initial line, the methionine production is a p p r o x i m a t e l y similar. Their b e h a v i o u r however cannot be generalised for each of t h e m shows a specific situation in relation to the respective parental m u t a n t s . Discussion

The empirical d a t a given in the present paper show t h a t each of the genes studied influences the synthesis Theuret. Appl. Ge~zetics, l'ol. 45, No. 1

of tile seed proteins in a distinguishing way. An interesting insight in the relations between the composition of the genome and the biosynthesis of metabolic p r o d u c t s serving as storage substances in the earliest ontogenetie stages is already o b t a i n e d from the analysis of a small n u m b e r of m u t a n t s . These results are of interest for m u t a t i o n breeding since i m p o r t a n t substances required for animal and h u m a n nutrition are involved. Moreover, the comparison of n m t a n t s and recombinants enables us to discuss the problenl of the combined action of nmtant.genes. 1. The Gene~ically Co~ditioned Variability o~ the Seed Proteins An interesting result of our investigations consists in the fact t h a t nearly each m u t a n t analysed has a specific p a t t e r n of seed proteins differing not only from the initial line but also from the other m u t a n t s . As shown b y Adriaanse et al. (1969) in different varieties of Phaseolus vulgaris these p a t t e r n s are characteristic and constant for each g e n o t y p e and are not influenced b y e n v i r o n m e n t a l factors. The genes in

18

W. Gottschalk and H. P. Miiller : Seed Proteins of Mutants and Recombinants of Pisum sativum

question do certainly not control the biosynthesis of the seed proteins directly, they influence these processes indirectly. Thus, the genes studied are not comparable with the voluminous group of genes controlling the course of meiosis and being directly responsible for the formation of funetionable germ cells (Gottschalk 1968, t973). It cannot be expected that it is possible to find the gene groups responsible for the synthesis of the seed proteins in radiation genetic experiments. Mutants of this type would represent biochemically deficient genotypes which are not viable because of the lack of single amino acids (Hess t 968). The findings given in the present paper could be interpreted b y means of the concept of the "biochemical pleiotropy". A distinct gene not concerning directly with the protein synthesis gets mutated. The gene products resulting from the m u t a t e d gene induce changes in the biosynthesis of other substances during the ontogenetic development. In this way, the gene indirectly can influence the composition of these substances from which the storage proteins of the seeds are studied in the present paper. Not only the changes of the quantitative situation of certain protein components but preferably the striking differences of the protein patterns demonstrate that the respective genes influence the protein metabolism on a broad base. It can be assumed, that not only the proteins but also other groups of substances are altered under the influence of the m u t a t e d genes. This could already be demonstrated for the five main sugars of the seeds of some of these m u t a n t s (Thierfeldt i972). I t is our opinion t h a t the pleiotropy of the gene action involves different biosyntheses of the organism the clarification of which requires comprehensive biochemical analyses. Another possibility for interpreting our findings would be the assumption of the action of modifying genes (Johnson et al., i 97(t). The co-operation of the genes studied which indirectly influence the protein synthesis is of particular interest. This can be demonstrated by comparing the recombinants with their parental mutants. The findings available from the genotypes analysed show, t h a t the total protein amount and the lysine production of the recombinants agree well with those of the low parental m u t a n t . In contrast to this, the methionine content of the recombinant resembles to that of the high parent (fig. 11). These findings obtained by means of the amino acid analyser were fully confirmed after having studied the same genotypes by microbiological methods (Henke 1972). However, these clear correlations could not be confirmed when we consider not only the protein content of equal amount of seed meal but also the seed production of the genotypes. It could be shown, that the combined action of specific genes has negative consequences with regard to the seed protein synthesis quantitatively as well as qualitatively. It is obvious, that the relations found in the small group of m u t a n t s and recombinants analysed in tile present investigations

cannot be generalized. On the contrary, each gene combination will result in a specific biochemical situation. 2. The Breeding Value o/ M u t a n l s and Reeombinants under Consideration o / t h e Seed Proteins

The findings of the present paper show that there are clear differences between the genotypes studied with regard to the quantitative and qualitative situation of the seed proteins. These data represent an essential supplement for the estimation of the breeding value of the respective strains considering their seed yield as well. Two examples are demonstrated to show how the biochemical situation of m u t a n t s can influence their qualification for breeding purposes. The useful character of m u t a n t 46C is its considerable earliness. In 1973, it was the only m u t a n t of a group of different genotypes which was able to seed in the arid areas of India, while other m u t a n t s of our collection as well as some Indian local varieties dried prematurely as a consequence of the lack of the monsoon rainfall. Thus, it has demonstrated its positive selection value under extreme clinratic conditions. In West Germany it is not of interest for pea breeding because of its low seed yield. In the protein content it does not differ from the initial line but it shows a negative shift between tile two main fractions; tile proportion of the globulins is increased, that of the albumins is decreased. This shift means a qualitative degradation of the proteins as the albumins represent the nutritionally more valuable fraction because of their amino acid composition (Bajaj et al. 1971, Boulter and Derbyshire 1971). This shift appears also with regard to the total amount of tile essential amino acids; it lies below the corresponding values of the initial line. The m u t a n t shows a more favourable situation than the initial line with regard to specific amino acids such as lysine and methionine. However, this increase does not represent a positive yield trait because the seed production of the m u t a n t is too low. Considering this criterion protein yields of only 75 to 8O~o of the control values are reached. The same relations are valid for the total content of the essential amino acids. Thus, the unfavourable yielding capacity is not compensated by a favourable quantity or quality of the seed proteins. Mutant 12olA shows an increased seed production because of its stem bifurcation. The protein content of its seed flour does not essentially differ from that of the initial line. The albumin fraction is somewhat reduced but the total amount of the essential amino acids is clearly increased than in the control. With the exception of phenylalanine, threonine and valine all the other essential amino acids are present in higher quantities. The protein yield per plant of this m u t a n t is 1o--3o~'o higher because of the high seed production. Also a qualitative improvement can be stated in addition to the quantitative one because Theoret. Appl. Ge~elics, l'ol. 45, No. 1

\V. G o t t s c h a l k ;tnd H. 1'. Mtiller: Seed l ' n , t c i n s of M u t a n t s and R c c o n l b i n a n t s of ]qsm~z saliwtm

19

t h e p r o d u c t i o n of e s s e n t i a l a m i n o a c i d s p e r p l a n t is l i k e w i s e i n c r e a s e d . T h u s , t h e f a v o u r a b l e s e e d y i e l d of n m t a n t 1201A is s u p p l e m e n t e d b y an a d v a n t a g e o u s q u a n t i t a t i v e a n d q u a l i t a t i v e a l t e r a t i ( m of t h e seed proteins.

Horn, M. J., Blum, A. C., Gersdorf, C h . E . F., \Varren, H. \V. : Sources of error in microbiological d e t e r m i n a tion of a m i n o acids on storage. General Chemistry, St. Paul, Minneapolis, Vol. 32, p. 34 (1955).

Literature

Hynes, M. J.: G e n e t i c a l l y controlled electrophoretic variants of storage protein in /~ salivum. Australian J. Hiol. Sci. 21, 827--829 (1968).

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W. G o t t s c h a l k and If. P. Mtiller : Seed Proteins of M u t a n t s and R e c o m b i n a n t s of P i s u m sativum

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Professor l)r. \V. G o t t s e h a l k Professor H. P. Miiller I n s t i t u t fiir Genetik der Universitttt B o n n Kirschallee 1 ])-53 B o n n ( G e r m a n y / B R D )

Theoret. A p p l . Genetics, Vol. 45, No. 1