- Email: [email protected]
Effect of the opaque and floury mutations on the accumulation of dry matter and protein fractions in maize endosperm
Plant Physiology and Biochemistry 43 (2005) 549–556 www.elsevier.com/locate/plaphy
Original article
Effect of the opaque and floury mutations on the accumulation of dry matter and protein fractions in maize endosperm Jacques Landry a,*, Catherine Damerval b, Ricardo A. Azevedo c, Sonia Delhaye a a
Inra, Laboratoire de Chimie Biologique, INA-PG, 78850 Thiverval-Grignon, France Station de Génétique Végétale, Inra/UPS/INA-PG/CNRS UMR8120, La Ferme du Moulon, Gif-sur-Yvette, France c Departamendo de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, SP, Brazil b
Received 17 January 2005; accepted 11 May 2005 Available online 04 June 2005
Abstract Grains of nine opaque (o) and floury (fl) mutants of maize (Oh43o1, Oh43o2, B79o5, B37o7, W22o10, W22o11, W22o13, Oh43fl1 and Oh43fl2) were examined for the weight proportions of their component tissues and the content of eight nitrogen fractions in their endosperms. A linear regression was found connecting the amounts (mg per endosperm) of zeins and true proteins (crude proteins minus non-protein nitrogen) for the non-opaque2 mutants. The data points connecting zeins to true proteins present in the mature endosperms of six wild-type (+) inbred lines and their o2 versions were located outside (+) or within (o2) the 95% confidence range of the regression line. The data obtained from the developing and mature endosperms of the W22o7 inbred line (Di Fonzo et al., Plant Sci. Lett., 1979, 77) and the floury portion of mature endosperms of three other wild-type inbred lines fell practically on the regression line. The effects of genotype and environmental factors upon the relative accumulation rate of zeins were assessed from the present results and the data taken from the literature concerning the quantitative interdependence between zeins and true proteins in immature and mature endosperms. © 2005 Elsevier SAS. All rights reserved. Keywords: Endosperm; Linear regression; Maize; Opaque and floury mutations; Zea mays L.; Zein and true protein accumulation
1. Introduction Seed proteins are important in animal nutrition and food processing [23]. The protein content of a maize grain is dependent upon the genotype and the growth conditions of the plant. Protein is customarily calculated from the total nitrogen (referred to as crude protein) or more rarely from the difference between total and non-protein nitrogen (NPN, referred to as true protein). The discrepancy between the protein values can vary markedly according to the genotype (wild-type or mutant), physiological age of the grain and the growth conditions. The true proteins can be differentiated by selective extraction into six fractions (F), which are grouped together into two classes designated as zeins and non-zeins. Non-zeins, formerly termed basic proteins [14] are made up of albumins plus globulins (FI) and true glutelins (FV + FVI), rich in lysine * Corresponding author. Fax: +33 1 30 81 53 73. E-mail address: [email protected] (J. Landry). 0981-9428/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2005.05.002
and tryptophan. Distributed throughout all tissues of the grain, they are present right the way through development, following pollination, since they are comprised of enzymes and other proteins important in metabolism. Zeins, formerly termed endosperm specific proteins [14], are made up of free a-subunits (FII), b-, d- and bound a-subunits (FIII) and c-subunits (FIV), and contain very low amounts of lysine and tryptophan. Located in the endosperm only and more specifically in protein bodies, they are synthesized for a period after pollination and consequently later than non-zeins [14,27]. Furthermore, a linear correlation has been established between the absolute amounts of zeins and true proteins accumulated in the developing endosperm originating from wild-type and mutant inbred lines [11]. The straight line has a slope corresponding to the relative rate of zein accumulation and is independent of growth conditions of the plant during development of the grain. The x-intercept provides an approximate value of the amount of non-zeins accumulated in the endosperm, prior to that of zeins [11]. Quantitatively, zeins make up 58% and 70% of the total nitrogen present in the mature
550
J. Landry et al. / Plant Physiology and Biochemistry 43 (2005) 549–556
grain and endosperm, respectively, of the commercial maize variety Inra 260 [14]. Such characteristics result in a deficiency in the grain of lysine and tryptophan, and are the cause of its poor nutritional quality, which is not improved by agricultural and breeding practices, since the proportion of nonzeins in protein decreases with increasing protein content. The discovery that the grain of mutants such as opaque-2 [19] and floury-2 [20] contain higher concentration of lysine than their wild-type counterparts (due to a reduction in the amount of zein in the endosperm), has opened the way for seeking other mutants displaying similar traits. The response of the endosperm following such mutations has been estimated by quantifying their zein or lysine content, together with that of crude proteins, the results being expressed on a dry matter or a crude protein basis, or more rarely on a per endosperm basis. These data are to be treated with caution, due to the underestimation of zeins caused by incomplete extraction, or to their overestimation originating from the presence of NPN in alcoholic extracts, and to the fact the level of NPN had not been determined. As a part of collaborative study concerning the influence of a range of opaque mutations on maize lysine metabolism, we have characterized endosperm samples from a biochemical standpoint, by analyzing the nitrogen fractions and by expressing the data on a dry matter basis [1–3]. In the present investigation, the same mutant samples were examined from a physiological standpoint, by determining the weight proportions of various tissues of the grain, together with the concentrations of eight nitrogen fractions expressed on a per endosperm basis. Zein concentrations were correlated with that of true proteins and compared with the corresponding values obtained from six wild-type inbred lines and their opaque-2 (o2) versions and from the floury (opaque) part of endosperms of eight other wild-type inbred lines. These comparisons have allowed a further insight into the physiological regulation of the accumulation of zein proteins in opaque endosperms.
2. Results 2.1. Grain weight and weight proportions of various endosperm tissues The grain weight, together with the weight proportions of endosperm, embryo and pericarp for each of mutant lines examined in this study, are shown in Table 1. It can be seen that there is: 1) a large variability in the grain weight, which can be more than 100% depending on the genotype. It is noteworthy that when compared to 21 wild-type lines grown under close growth conditions, the mutants Oh43o1, B37o7, Oh43fl2 and W22o13 have grain of similar weight, whereas others have lighter grains (data not shown); 2) an independence of the proportion of pericarp in relation to genotype and grain weight; 3) a lower weight proportion of endosperm compensated by a higher proportion of the embryo in the case of the Oh43o2 line; and 4) an independence of weight proportion of the endosperm and embryo for non-o2 genotypes, the proportions being similar to those observed for 21 wild-type lines. 2.2. Distribution of nitrogen fractions To better characterize proteins from the mutants, we have isolated nine nitrogen fractions, namely: free amino acids (FAA), NPN, albumins + globulins, alcohol-soluble zeins (E3), pH 10 buffer-soluble zeins (E4), total zeins, true glutelins, non-zeins (NZ) and total true proteins. Table 2 lists the amounts of these different fractions in the endosperm, together with those of crude protein, and percentage of crude protein in the dry matter. The amounts of the same fractions originating from three wild-type lines Oh43+, W22+ and B37+, used as background for the mutations are also listed in Table 2 for comparison. Comparisons of the fraction amounts in the various genotypes were done by analyses of variance, and multiple comparisons of means using the Bonferroni correction with ␣ = 0.05 (not shown). The following results emerge: 1) the three wild-type lines are similar for salt-soluble nitrogen
Table 1 Grain weight and weight proportions of the principal tissues of the maize grain according to genotype Genotype Oh43o1 Oh43o2 B79o5 B37o7 W22o10 W22o11 W22o13 Oh43fl1 Oh43fl2 (non-o2) a,b 21 lines + a,c a
Wg (mg per grain) 210 121 128 209 125 102 149 135 190 156 ± 42 232 ± 35
Wendosperm/Wg (%) 79.4 70.6 81.2 82.1 83.4 82.2 77.3 83.0 80.8 82.3 ± 1.0 81.8 ± 2.2
Mean ± standard deviation. Opaque and floury mutants other than o2. c Wild-type lines selected for their differences in vitreousness.
b
Wgerm/Wg (%) 13.3 21.3 13.5 10.9 8.7 10.2 15.1 9.9 12.9 11.7 ± 2.3 11.0 ± 1.3
Wpericarp/Wg (%) 7.2 8.0 5.2 7.0 8.0 7.6 7.4 7.0 6.4 7.0 ± 0.9 7.2 ± 1.4
J. Landry et al. / Plant Physiology and Biochemistry 43 (2005) 549–556
551
Table 2 Distribution of nitrogen compounds in the maize endosperm according to genotype (data expressed as mg protein per endosperm) Genotype Oh43o1 Oh43o2 B79o5 B37o7 W22o10 W22o11 W22o13 Oh43fl1 Oh43fl2 Oh43+ B37+ W22+
FAA a 0.16 0.12 0.21 0.22 0.56 0.23 0.14 0.20 0.23 0.13 0.30 0.31
NPN b 0.67 0.36 0.47 0.56 0.89 0.55 0.38 0.39 0.66 0.30 0.50 0.60
E1,2 c 1.78 1.71 1.46 2.21 1.63 1.22 1.62 1.11 2.27 0.63 1.19 1.30
A+Gd 1.11 1.35 0.99 1.65 0.74 0.67 1.24 0.72 1.61 0.33 0.69 0.70
E3 e 18.17 4.56 14.05 16.21 7.84 7.40 10.93 13.72 15.03 12.25 15.07 17.03
E4 f 0.94 0.53 0.74 0.89 0.67 0.55 0.75 0.60 0.71 0.60 0.70 0.80
Zeins g 19.11 5.09 14.79 17.10 8.51 7.95 11.68 14.32 15.74 12.85 15.77 17.83
E5,6 h 6.97 5.46 5.32 7.01 3.87 4.96 5.59 4.49 6.26 3.00 4.35 5.10
NZ i 8.08 6.80 6.31 8.65 4.61 5.62 6.59 521 7.87 3.33 5.04 5.80
REi j 27.85 12.25 21.57 26.31 14.01 14.12 18.65 19.92 24.27 16.48 21.31 24.23
RtP k 27.19 11.89 21.10 25.75 13.12 13.57 18.27 19.53 23.61 16.18 20.81 23.63
P/DM% l 11.6 8.7 14.8 11.1 9.8 12.1 11.1 12.8 11.2 10.8 13.1 13.3
a
FAA: free amino acids (crude TCA extract). NPN: non-protein nitrogen (hydrolyzed TCA extract). c E1,2: salt extract (0.5 NaCl, 4 °C). d A + G: albumins + globulins (E1,2–NPN). e E3: alcohol extract. f E4: pH 10 buffer extract (water-soluble zeins). g Zeins: E3 + E4. h E5,6.: residue: true glutelins. i NZ: non-zeins: true proteins–zeins. j REi: crude proteins. k RtP: true proteins (REi–NPN). l Crude protein content in dry matter (DM). b
(E1,2) and albumins + globulins, but statistically different for E3, E4 and E5,6, 2) compared to wild-type lines, o11, fl1, o10, o13 and o5 have the same amount of salt-soluble nitrogen, while fl2, o1, o2 and probably o7 are richer; 3) o10, fl1, o11 and o5 have an albumin + globulin content similar to that of wild-type lines while fl2, o2, o13, o7 and probably o5 are richer, the highest content being shown for o7; 4) the level of zeins, compared to that of Oh43+, is not different for fl1 and fl2, but it is significantly higher for o1 and lower for o2; it is lower for o13 and for o11 and o10, when compared to that of W22+; 5) regarding the amounts of non-zeins the following hierarchy is highlighted: o1 ≅ fl2 > o2 > fl1 > Oh43+; further, o7 and o13 have higher amounts than their corresponding wild-type lines, while o10 has lower amount and o11 is similar to W22+; 6) regarding amounts of true proteins the hierarchy is: o1 > fl2 > fl1 > Oh43+> o2; further, o7 and o13 are not significantly different from their wild-type counterparts, but o10 and o11 have lower amounts than W22+. On the other hand a slightly different picture is seen when the data are expressed as a percentage of total crude proteins (data not shown). Thus zeins range from 41.5 (o2) to 71.9% (fl2), increasing to 68.6% in o1 only. These percentages are lower than those found for the wild-type lines (74.8% on average). Finally the hierarchy of the magnitude of total crude protein varies according to the basis (per endosperm or dry matter) used for expressing the data. 2.3. Relationship between zeins and true proteins A closer examination of the data presented in Table 2, brings out a parallelism between the quantitative variations of zeins and those of true proteins among the mutants.
To further investigate the effect of the opaque mutation on the accumulation of zein in the endosperm, the amount of zein expressed per endosperm (WZ) has been plotted against the amount of true protein (WtP) for all the nine mutants used in this study. A linear correlation is observed (Fig. 1) for the eight non-o2 mutants, with a regression line for the equation: WZ = –(1.62 ± 1.13) + (0.752 ± 0.054) WtP of r2 = 0.970 (1). On the other hand, the two parallel lines, each having a ver-
Fig. 1. Total zeins (E3 + E4) in whole endosperms of all lines used in the study: opaque maize (.), wild-type inbred lines (*) and their o2 versions (C). The regression line is relative to non-o2 mutants; and the underlined data points to Oh43o2 inbred lines. Data from present study (.) and from Landry et al. [16] (*, C).
552
J. Landry et al. / Plant Physiology and Biochemistry 43 (2005) 549–556
tical distance 2.31 sˆyx = 1.73 mg, constitute the 95% confidence limits. The data point for Oh43o2 is seen to be outside of the lower limit. The regression line related to the zein accumulation in the mature endosperm of six wild-type lines, taken as reference, has the equation: WZ = –(0.062 ± 0.061) + (0.792 ± 0.029) WtP with r2 = 0.987 (2). It is parallel to the above one, as the slopes are not significantly different, and merges practically with the upper 95% confidence limit, which shows the absence of discontinuity between wild-type and mutants, regarding the levels of zeins and true proteins in the endosperm. In contrast, the data points concerning zein accumulation in the endosperm of six o2 versions of these inbred lines, including the Oh43o2 genotype, are scattered in confidence range, which provides evidence that the regression line determined for non-o2 mutants can also be fitted for o2 ones. The discrepancy recorded between the Oh43o2 sample characterized in the present study and that examined previously [15,16] for zein and true protein content, highlights the intragenotype variability in the distribution of nitrogen fractions. To demonstrate the general character of this regression line, we have also included data previously published by Di Fonzo et al. [8]. These authors determined the effect of the o7 mutation on the distribution of six nitrogen fractions (NPN, FI....FV) in the developing endosperm of W22o7. At maturity, the endosperm contained 7.5 mg of true proteins and 3.7 mg of zeins (FII + FIII + FIV). The absence of any significant discrepancy between these experimental values and those deduced from the regression line (WZ = 4 mg for WtP = 7.5 mg), allows six values obtained from the data of Di Fonzo et al. [8] (five of which correspond to immature endosperms), to be introduced into the new regression, which has the following equation: WZ = –(2.21 ± 0.35) + (0.779 ± 0.021) WtP with r2 = 0.980 (3). On the other hand, the two parallel lines, each having vertical distances 2.2 sˆyx = 1.3 mg, delineate a narrower 95% confidence range (Fig. 2). This regression represents the average variations of zein accumulation in developing and mature endosperms of non-o2 mutants. The data points around the wild-types inbred lines are seen to be excluded from the regressions, whereas those around their o2 versions belong to it, confirming the above conclusions. Maize grains with the opaque phenotype have endosperms with a soft (floury) texture only. Some similarities between the floury portion of wild-type endosperms and the o2 endosperm have been reported as for the appearance of starch granules, the limited number and size of protein bodies, and the distribution of nitrogen fractions [17,21]. We have attempted to establish whether these similarities can be revealed by the relationship connecting zein and true protein content, expressed per endosperm. In this context, the amount of zein present in the floury region of endosperms originating from 8 wild-type inbred lines has been plotted against the corresponding true protein content (Fig. 2). The zein content of floury endosperms, the richest in true proteins (three samples), is able to fit the regression (3) defined for non-o2 samples. The data points of the three samples with a medium
Fig. 2. Total zeins (E3 + E4) in whole endosperms of opaque maize (.), wild-type inbred lines (*) and their o2 versions (C), W22o7 inbred line (D) and in the floury portion of mature endosperms from wild-type inbred lines (e). Data points (D) the lowest in zeins, correspond to immature stages. The regression line is relative to non-o2 and W22o7 maize. The data point (.) outside the 95% confidence range, corresponds to Oh43o2. Data from present study (.), from Landry et al. [16] (*, C), from Di Fonzo et al. [8] (D) and from Landry et al. [17] (e).
content of true proteins are located at the upper 95% confidence limit of the regression, the two others being outside. For a high level of true proteins there is a quantitative similarity between the relative contents of zeins in floury portion of endosperm of wild-type lines and in the whole endosperm of mutant lines. 3. Discussion 3.1. Estimation of zein content In the present study, zeins have been considered as the proteins isolated in extracts E3 and E4. E4 is a minor (2.9–4.8% of the total nitrogen) fraction, which is ill-defined when it is subjected to polyacrylamide gel electrophoresis in the presence of SDS. E4 has an amino acid composition intermediary between those of zeins and true glutelins, but the lysine content is closer to that of zein than that of true glutelin [17]. A good agreement was reported between the endosperm content in FII + FIII + FIV (or E3 + E4) determined experimentally and that estimated from the lysine content of the endosperm by assuming a lysine content of zero for zeins and 7% for non-zeins [12]. Furthermore, the (E3 + E4)/E3 ratio calculated from the data of Table 2 for non-o2 mutants, has an average value of 1.06 ± 0.01. Assuming that only half of the proteins present in E4 are zeins, leads to an overestimation of 3%. This is lower, if small traces of zeins are present in the residue (FVI). The protein content of E3 is also given in Table 2 for comparison with other works dealing with the content of alcohol-soluble zeins only (see below). 3.2. Relationship between zeins and true proteins of non-o2 opaque mutant endosperms The linear relationship Wz = a + bWtP connecting zein and true protein content of endosperm, is characterized by two
J. Landry et al. / Plant Physiology and Biochemistry 43 (2005) 549–556
parameters: the slope b and the y-intercept a. The slope b, defined as relative rate of zein accumulation, has been shown to be independent of growth conditions but dependent of genotype [11]. In the present case the slope b is found to be practically independent of genotype, wild-type or non-o2 opaque mutant, revealing a similarity in the relative rate of zein accumulation. Thus, under the growth conditions used for obtaining the samples analyzed in the present study, approximately 0.78 mg is synthesized as zeins, when the wild-type or mutant endosperms accumulate 1 mg of true protein. The y-intercept a, which is negative, allows an approximation of the magnitude of the x-intercept (–a/b). Since b is considered as constant, the absolute value of a is proportional to the x-intercept, thus the value of the latter gives an estimate of the level of non-zeins accumulated prior to zein synthesis. This value may be a slight overestimation, since zein synthesis starts progressively and not abruptly as it is depicted in Fig. 2. Under the growth conditions used in the present study, the endosperms of non-o2 mutants with respect to those of the wild-type, synthesize a comparable or greater amount of non-zeins, before synthesizing zeins at a similar relative rate, the final amount of these proteins being dependent on the genotype. The same process would be also true for the floury portion of wild-type endosperm, when compared with that occurring for horny portion of the flouriest endosperms, in agreement with a strict control of the spatial and temporal expression of the zein genes [7]. The present data bring out a great variability in zein content with the introduction of a given mutant gene in different wild-type backgrounds. This is particularly marked, when W22o7 is compared with B37o7 (see Fig. 2) or Oh 43fl2 (W Z = 15.7 mg for W tP = 23.6 mg) with R807fl2 (WZ = 7.6 mg for WtP = 14.8 mg as calculated from the data of Sodek and Wilson [25]). In other words, a description of the effect of a mutant gene on the accumulation of zeins, without specifying the wild-type background in which it has been introduced, can only be incomplete. Interestingly, the endosperm content of zeins and true proteins of R807fl2 is similar to those found for W22o10 or W22o11. A confirmation of the above statement is provided by analyzing the data reported by Balconi et al. [4]. In a study similar to this, the investigators determined the protein distribution (FI, FII + FIII & FIV + FV + FVI) in developing (two stages) and mature endosperms of nearly isogenic inbred lines of A69y+, o1, o2, o9, o11, fl1 and fl3. Fig. 3 compares the data of Balconi et al. [4] and that reported here, for the amounts of zeins (FII + FIII) and (E3) per endosperm, plotted against the amount of total true proteins. A noteworthy similarity and a marked linear relationship can be seen. The regression line involving all data points including those relative to o2 mutant but not those pertaining to the earliest stage of development (because at this stage the relative rate of zein accumulation is still increasing and has not yet reached its definitive and constant value characterizing the later stages) has for equation: WZ = –(1.72 ± 0.46) + (0.742 ± 0.025) WtP with r2 = 0.982 [4].
553
Fig. 3. Zeins (E3) in mature endosperms isolated from the grains of opaque maize (.) and in the developing and mature endosperms from the wild-type A69y inbred line (*) and the corresponding o2 (C), o1, o9, o11, fl1 (n) and fl3 (M) versions. The regression line is relative to the maize lines originating from A69y. Data points outside the 95% confidence range correspond to A69yo2 and Oh43o2. Data from present study (.) and from Balconi et al. [4] (*, C, n and M).
Several observations can be made when the data of Balconi et al. [4] are compared with the present results: 1) the slopes of regression lines (2) and (4) are not significantly different. The same is true of the relative rate of zein accumulation, suggesting practically identical growth conditions prior to pollination. However, the ratio of these slopes amounts to 1.05 and is similar to the mean of the ratios (E3 + E4)/E3 calculated above (1.06), agrees with the significant discrepancy due to the presence of proteins present in the E4 extract; 2) the data points relative to o2 mutants are located at the lower 95% confidence limit, suggesting a major accumulation of non-zeins prior to that of zeins, or a reduction the rate of zein accumulation; 3) fl3 exhibits the same behavior as the other non-o2 mutants, the low content in zeins paralleling that of the true proteins; 4) the regression equation (4) includes the values related to the wild-type line A69y+, providing further evidence for the lack of discontinuity between wild-type and mutant samples, regarding the amounts of zeins and true proteins expressed per endosperm. In contrast, the lysine content of the crude proteins of A69yo1 (4.2%), as determined by Balconi et al. [4] using a colorimetric method, is inconsistent with that estimated as 1.9% from the zein content of true proteins [12]. This latter value is close or identical to that reported for the crude proteins of W64Ao1 by Mertz [18] (1.7%), and by Hunter et al. [10] (1.9%, by taking into account of conversion factor of 5.7), indicating that A69yo1 cannot be considered as a highlysine genotype. 3.3. Relationship between zeins and true proteins of opaque-2 endosperms The present data, together with those taken from the literature suggest a process of zein accumulation in the o2
554
J. Landry et al. / Plant Physiology and Biochemistry 43 (2005) 549–556
Fig. 4. Zeins (E3) in immature (D, a, M, e) and mature (D, a, M, e, C, ., m, ▼) endosperms isolated from the grains of opaque-2 maize: W64Ao2 (D) from Soave et al. [24], (a) from Wall and Bietz [26], B37o2 (M) from Salamini et al. [22], A69yo2 (e) from Balconi et al. [4], o2 inbred lines (C) including Oh43o2 (underlined) and B37o2 (double underlined) from Landry et al. [15]; Oh43o2 (.) from present study; Inrao2 (m) from Landry and Moureaux [14]; and R802o2 (▼) from Sodek and Wilson [25]. For each of genotypes D, a, M, e, C the mature state corresponds to the data point located the most to the right.
endosperm slightly different from that described above for other mutants. In fact, some data points concerning the o2 mutants are sometimes outside, or near to the lower 95% confidence limit of the regression lines. To better assess the effect of the o2 gene upon zein accumulation, it is necessary to know the relationship between zeins and true proteins in immature o2 endosperms. In this context we have analyzed some data taken from the literature [22,24,26] as depicted in Fig. 4. This analysis brings out two processes of zein accumulation, which leads practically to the same final level of zeins in the endosperm. In the first case [22,26] zeins are synthesized continuously until maturity with relative rates (0.405 for W64Ao2 and 0.490 for B37o2) that are lower than those recorded for wild-type backgrounds (0.803 for W64A+ and 0.611 for B37+), grown under the same conditions. In the second case [24], two phases are observed for the zein accumulation in W64Ao2. The first is characterized by zein synthesis at a high relative rate (0.706), close to that found for non-o2 mutants (0.735), the second by the synthesis of zeins at a very low rate and non-zeins at a high rate. It is noteworthy that the relative rate of 0.706 is higher than that (0.599) found for the wild-type background, grown under different conditions [11] and it is the only example in which the relative accumulation is observed to take place in two phases. The fact that two processes of zein accumulation are seen for the same genotype, reveals the impact of different growth conditions. The cessation of zein synthesis could be related to the failure of protein bodies to store them, which could promote the synthesis of non-zeins by a compensatory effect in some cases, but it could also result from the cessation of protein synthesis, as suggested by the data from Habben et al. [9]. The latter authors reported the same lysine content in W64Ao2 endosperm at mid-maturity as maturity, providing
Fig. 5. Zeins (E3) in immature and mature endosperms isolated from the grains of opaque maize: W64Ao2 (D) from Soave et al. [24], (a) from Wall and Bietz [26]; W22o7 (▼) from Di Fonzo [8]; and A69Yfl3 (m) from Balconi et al. [4]. For each of genotypes the mature state corresponds to the point located the most to the right. Note that the data point relative to maturity for A69Yfl3 (m) corresponds to that relative to W64Ao2 (D) at midmaturity (data point preceding that located the most to the right).
evidence that the non-zeins were not synthesized during the second phase of maturation. In fact, plotting the zein content against the true protein content in the mature endosperms of various o2 genotypes, gives a scatter diagram highlighting a marked effect of genotype and growth conditions on the relative rate of zein accumulation. Thus, the relative rate of zein accumulation would vary from 0.40 to 0.71. A variability of the same amplitude (from 0.43 to 0.80) is seen for this parameter when Illinois Low Protein is compared to Illinois High Protein [11]. It is noteworthy that the data points from Wall and Bietz [26] for W64Ao2, from Salamini et al. [22] for B37o2, from Sodek and Wilson [25] for R802o2 and from Landry [12] for Inrao2 are on the outside of the lower 95% confidence limit, indicating an effective reduction in the relative rate of zein accumulation in respect to that observed with the wild-type backgrounds. The actual efficiency of the o2 mutation to reduce zein accumulation may be seen only with some samples, the conditions for obtaining the reduction, being ill defined. This variation could explain the lack of precise conclusions about the action of the o2 gene upon zein accumulation, despite numerous studies. On the other hand, the amount of non-zeins accumulated prior to zein synthesis, ranges from 2.2 and 2.9 mg per endosperm for any mutant line, as deduced from the value of the x-intercept of the regression lines. For eight wild-type lines selected among 21 ones for their differences in vitreousness (Table 1 and [17]) the amount of non-zeins was found to vary from 0.3 (for the horniest endosperms) to 2.7 mg (for the flouriest endosperms) (Landry, unpublished data). Therefore, a higher level of non-zeins in the endosperm prior to the zein synthesis, is not a characteristic specific to the presence of the opaque gene but to a floury nature. From the foregoing, the conclusions drawn about the accumulation of zeins in the endosperm of non-o2 mutants remain valid in the case of o2 mutation, as can be seen from the data depicted in Fig. 5. Thus, the levels of zeins and true proteins
J. Landry et al. / Plant Physiology and Biochemistry 43 (2005) 549–556
in the mature endosperms of mutant line A69yfl3 are seen to be approximately the same as those found in the W64Ao2 endosperm at mid-maturity by Soave et al. [24], indicating a reduction in metabolism by 50%. In the case of mature endosperm of W22o7 these same levels are identical to those reported by Wall and Bietz [26] for mature endosperm of W64Ao2. However, the o2 mutation would differ from non-o2 opaque mutations, as the amount of zeins accumulated in the mature endosperm is reduced, irrespective of nature of the wild-type background. A similar quantitative reduction of zeins in the mature endosperm is also observed with the introduction of the o7 and fl2 genes, but such an alteration would be limited to some genetic combinations. 3.4. Molecular basis for the opaque mutation To our knowledge, the molecular basis for the opaque mutation is known for o2 and fl2 only. The O2 gene encodes a transcriptional activator that regulates expression of 22 kDa a-zein and 14 kDa b-zein genes as well as other genes not directly implicated in zein synthesis, including enzymes involved in amino acid and carbohydrate biosynthesis [1–3,6,9,10]. Almost all o2 mutants characterized have an altered O2 protein, promoting alteration in zein accumulation and in endosperm metabolism. These large pleiotropic effects may be involved in the particular relationships between zeins and true proteins, as compared to other mutants. The fl2 mutation has originated from a defective signal peptide of a 22 kDa a-zein, thus altering its processing [5]. Many other endosperm mutants have been reported to have opaque phenotype. These includes a series of the so classified opaque and floury mutants. There is no evidence that the genes encoding these phenotypes has any to do with transcription factors. They do alter physical, chemical and metabolic properties of the endosperm. The work described here is an attempt to connect all these mutants to a concerted endosperm developmental and metabolic chart.
555
types grown under various conditions. The same also holds for zeins from high-lysine genotypes. The high-lysine content is related to a reduction of the true protein level, and thereby of zein level. In some cases there would be a relative rate of zein accumulation, lower than those recorded for wildtype inbred lines.
5. Methods 5.1. Plant material The maize (Zea mays L.) genotypes Oh43o1, Oh43o2, B79o5, B37o7, W22o10, W22o11, W22o13, Oh43fl1 and Oh43fl2 were grown in a glasshouse in the field station of the Genetics Department, Piracicaba, University of São Paulo (Brazil) during 1999–2000 summer season. The wild-type (+) and opaque-2 (o2) versions of the six following inbred lines: W64A, W22, W23, Oh43, F2 and B37, were grown in a glasshouse at Orsay near Paris (France) in 1994 and 1995, (W23+ and o2) were also grown as references. All genotypes but F2 and F2o2 were obtained from the Maize Genetics Corporation Stock Center (University of Illinois, UrbanaChampaign, IL). Eight wild-type inbred lines (COEST6, F113, A188, CO255, LH74, CM105, F7 and ARGL256) selected for their differences in vitreousness, were grown in a glasshouse in Orsay during 2000 and were used for the isolation of the opaque portion of their endosperms. Endosperms, germs and pericarps were isolated by hand dissection of five to eight grains of each line, previously soaked in water for 30 min. Opaque (floury) and horny (vitreous) fractions were isolated from whole endosperm using an adjustable speed grinding wheel [17]. After lyophilization, the fractions were weighed and the dry matter content determined. The endosperms were ground using a ball mill and the dry flour was defatted with hexane, prior to extraction [17]. 5.2. Extraction and quantification of nitrogen compounds
4. Conclusion To conclude, expressing the amount of true proteins on a per endosperm basis is an appropriate measure of the synthetic capacity of the endosperm. Plotting the amount of zeins per endosperm against the amount of true proteins per endosperm, allows the integration of the present data with those already reported in the literature (when they are exploitable) on the same graph and to extract the essential facts concerning the accumulation of zeins in the maize endosperm. The changes in zein amount as a function of true protein, are depicted by: 1) a linear plot in the case of a given genotype, or a set of close genotypes grown under similar conditions. The high values of the coefficient of determination, indicate not a functional relationship but the fact that zeins constitute the major part of true proteins; 2) a set of linear plots forming a scattered plot when these changes pertain to several geno-
Duplicate extracts (E) were obtained by making successive extractions of the endosperm flour with: 1) 0.5 M NaCl, then water (E1,2); 2) water (discarded); 3) 55% (w/w) isopropanol + 0.02% (w/v) dithiothreitol (DTT) (E3); 4) 0.5 M NaCl + 0.02% DTT buffered at pH 10 with sodium borate and sodium hydroxide (E4). The remaining residue (E5,6) contained proteins that could be extracted by SDS (E5) plus insoluble proteins (E6). NPN (E0 by extension) was isolated from precipitated proteins by adding trichloroacetic acid (TCA) to E1,2 extracts, to a final concentration of 10% (w/v). No significant differences have been found between E0 extracted with TCA directly or isolated after protein precipitation [15]. NPN and protein were quantified by the ninhydrin assay of a-NH2 groups (and ammonia) liberated after sample hydrolysis, using an equimolar mixture of 17 amino acids and ammonium sulfate (Pierce) as a standard and a conver-
556
J. Landry et al. / Plant Physiology and Biochemistry 43 (2005) 549–556
sion factor of 1.06 µg of protein for 10 nmol of amino acids. FAA were quantified from TCA solutions without preliminary hydrolysis. NPN and proteins from E0, E1,2 and E4 extracts were hydrolyzed in 3 M NaOH for 45 min [13]. Proteins from flour, E3 extract (after removing solvent) and residue (E5,6) were hydrolyzed in constant boiling HCl at 115 °C for 18 h. From the foregoing NPN, albumins plus globulins, zeins and true glutelins corresponded to a-amino nitrogen contained in E0, E1,2 – E0, E3 + E4, E5,6, respectively.
[7]
5.3. Statistical analysis
[11]
The method of least squares was used for determining the equations of regression together with the 95% confidence range from the standard error of estimate and the number of degrees of liberty. Comparisons of fraction amounts were done using oneway analyses of variance with the genotype as the factor. When a significant F value was obtained, multiple comparisons of means were done with the Bonferroni correction, with ␣ = 0.05.
[8]
[9]
[10]
[12] [13]
[14]
[15]
[16]
Acknowledgements The authors would like to thank Professor Peter Lea, Lancaster University, UK, for reviewing the manuscript and helpful suggestions.
[17]
[18]
[19]
References [1]
[2]
[3]
[4]
[5]
[6]
R.A. Azevedo, C. Damerval, J. Landry, P.J. Lea, C.M. Bellato, L.W. Meinhardt, M. Le Guilloux, S. Delhaye, A.A. Toro, S.A. Gaziola, B.D.A. Berdejo, Regulation of maize lysine metabolism and endosperm protein synthesis by opaque and floury mutations, Eur. J. Biochem. 270 (2003) 4898–4908. R.A. Azevedo, C. Damerval, P.J. Lea, J. Landry, C.M. Bellato, L.W. Meinhardt, M. Le Guilloux, S. Delhaye, A.A. Toro, S.A. Gaziola, V.A. Varisi, P.L. Gratão, Genetic control of lysine metabolism in maize endosperm mutant, Funct. Plant Biol. 31 (2004) 339–348. R.A. Azevedo, P.J. Lea, C. Damerval, J. Landry, C.M. Bellato, L.W. Meinhardt, M. Le Guilloux, S. Delhaye, V.A. Varisi, S.A. Gaziola, P.L. Gratão, A.A. Toro, Regulation of lysine metabolism and endosperm protein synthesis by the Opaque-5 and Opaque7 mutation, J. Agric. Food Chem. 52 (2004) 4865–4871. C. Balconi, N. Berardo, A. Reali, M. Motto, Variation in protein fractions and nitrogen metabolism of developing normal and opaque endosperm mutants of maize, Maydica 43 (1998) 195–203. C.E. Coleman, M.A. Lopes, J.W. Gillikin, R.S. Boston, B.A. Larkins, A defective signal peptide in the maize high-lysine mutant floury-2, Proc. Natl. Acad. Sci. USA 92 (1995) 6828–6831. C. Damerval, M. Le Guilloux, Characterization of novel proteins affected by the o2 mutation and expressed during endosperm development, Mol. Gen. Genet. 257 (1998) 354–361.
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
S. Facio Dolfini, M. Landoni, C. Tonelli, L. Bernard, A. Viotti, Spatial regulation in the expression of structural and regulatory storageprotein genes in Zea mays endosperm, Dev. Genet. 13 (1992) 264– 276. N. Di Fonzo, E. Gentinetta, F. Salamini, Action of the opaque-7 mutation on the accumulation of storage products in maize endosperm, Plant Sci. Lett. 22 (1979) 77–88. J.E. Habben, A.W. Kirleis, B.A. Larkins, The origin of lysinecontaining proteins in opaque-2 maize endosperm, Plant Mol. Biol. 23 (1993) 825–838. B.G. Hunter, M.K. Beatty, G.W. Singletary, B.R. Hamaker, B.P. Dilkes, B.A. Larkins, R. Jung, Maize opaque endosperm mutations create extensive changes in patterns of gene expression, Plant Cell 14 (2002) 2591–2612. J. Landry, A linear model for quantitating the accumulation of zeins and their fractions (a + d, b and c) in developing endosperm of wild-type and mutant maizes, Plant Sci. 163 (2002) 111–115. J. Landry, Quantitation of zeins from the lysine content of grain, endosperm and glutelins from maize, Maydica 48 (2003) 39–54. J. Landry, S. Delhaye, A simple and rapid procedure for hydrolyzing minute amounts of proteins with alkali, Anal. Biochem. 243 (1996) 191–194. J. Landry, T. Moureaux, Distribution and amino acid composition of protein groups located in different histological parts of maize grain, J. Agric. Food Chem. 28 (1981) 1186–1191. J. Landry, S. Delhaye, C. Damerval, Improved method for isolating and quantitating a-amino nitrogen as nonprotein, true protein, saltsoluble proteins, zeins and true glutelins in maize endosperm, Cereal Chem. 78 (2000) 620–628. J. Landry, S. Delhaye, C. Damerval, Effect of opaque-2 gene on accumulation of protein fractions in maize endosperm, Maydica 47 (2002) 59–66. J. Landry, S. Delhaye, C. Damerval, Protein distribution pattern in floury and vitreous endosperm of maize grains, Cereal Chem. 81 (2004) 153–158. E.T. Mertz, Genetic and biochemical control of grain protein synthesis in normal and high lysine cereals, World Rev. Nutr. Diet. 48 (1986) 222–262. E.T. Mertz, L.S. Bates, O.E. Nelson, Mutant gene that changes protein composition and increases lysine content of maize endosperm, Science 145 (1964) 279–280. O.E. Nelson, E.T. Mertz, L.S. Bates, Second mutant gene affecting the amino acid pattern of maize endosperm proteins, Science 150 (1965) 1469–1470. J.L. Robutti, R.C. Hoseney, C.E. Wassom, Modified opaque-2 endosperm. II. Structure viewed with a scanning electron microscope, Cereal Chem. 51 (1974) 173–180. F. Salamini, N. Di Fonzo, E. Fornasari, E. Gentinetta, R. Regiani, C. Soave, C. Mucronate, Mc, a dominant gene of maize which interacts with opaque-2 to suppress zein synthesis, Theor. Appl. Genet. 65 (1983) 123–128. P.R. Shewry, F. Beaudoin, J. Jenkins, S. Griffiths-Jones, E.N. Mills, Plant protein families and their relationships to food allergy, Biochem. Soc. Trans. 30 (2002) 906–910. C. Soave, F. Pioli, A. Viotti, F. Salamini, P.G. Righetti, Synthesis and heterogeneity of endosperm proteins in normal and opaque-2 maizes, Maydica 28 (1975) 83–94. L. Sodek, C.M. Wilson, Amino acid composition of proteins isolated from normal, opaque-2 and floury-2 corn endosperm by a modified Osborne procedure, J. Agric. Food Chem. 19 (1971) 1144–1150. J.S. Wall, J.A. Bietz, Differences in corn endosperm proteins in developing seeds of normal and opaque-2 corn, Cereal Chem. 64 (1987) 275–280. C.M. Wilson, Proteins of the kernel, in: S.A. Watson, P.E. Ramsted (Eds.), Corn: Chemistry and Technology, American Association of Cereal Chemists, St Paul, MN, 1987, pp. 273–310.
Original article
Effect of the opaque and floury mutations on the accumulation of dry matter and protein fractions in maize endosperm Jacques Landry a,*, Catherine Damerval b, Ricardo A. Azevedo c, Sonia Delhaye a a
Inra, Laboratoire de Chimie Biologique, INA-PG, 78850 Thiverval-Grignon, France Station de Génétique Végétale, Inra/UPS/INA-PG/CNRS UMR8120, La Ferme du Moulon, Gif-sur-Yvette, France c Departamendo de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, SP, Brazil b
Received 17 January 2005; accepted 11 May 2005 Available online 04 June 2005
Abstract Grains of nine opaque (o) and floury (fl) mutants of maize (Oh43o1, Oh43o2, B79o5, B37o7, W22o10, W22o11, W22o13, Oh43fl1 and Oh43fl2) were examined for the weight proportions of their component tissues and the content of eight nitrogen fractions in their endosperms. A linear regression was found connecting the amounts (mg per endosperm) of zeins and true proteins (crude proteins minus non-protein nitrogen) for the non-opaque2 mutants. The data points connecting zeins to true proteins present in the mature endosperms of six wild-type (+) inbred lines and their o2 versions were located outside (+) or within (o2) the 95% confidence range of the regression line. The data obtained from the developing and mature endosperms of the W22o7 inbred line (Di Fonzo et al., Plant Sci. Lett., 1979, 77) and the floury portion of mature endosperms of three other wild-type inbred lines fell practically on the regression line. The effects of genotype and environmental factors upon the relative accumulation rate of zeins were assessed from the present results and the data taken from the literature concerning the quantitative interdependence between zeins and true proteins in immature and mature endosperms. © 2005 Elsevier SAS. All rights reserved. Keywords: Endosperm; Linear regression; Maize; Opaque and floury mutations; Zea mays L.; Zein and true protein accumulation
1. Introduction Seed proteins are important in animal nutrition and food processing [23]. The protein content of a maize grain is dependent upon the genotype and the growth conditions of the plant. Protein is customarily calculated from the total nitrogen (referred to as crude protein) or more rarely from the difference between total and non-protein nitrogen (NPN, referred to as true protein). The discrepancy between the protein values can vary markedly according to the genotype (wild-type or mutant), physiological age of the grain and the growth conditions. The true proteins can be differentiated by selective extraction into six fractions (F), which are grouped together into two classes designated as zeins and non-zeins. Non-zeins, formerly termed basic proteins [14] are made up of albumins plus globulins (FI) and true glutelins (FV + FVI), rich in lysine * Corresponding author. Fax: +33 1 30 81 53 73. E-mail address: [email protected] (J. Landry). 0981-9428/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2005.05.002
and tryptophan. Distributed throughout all tissues of the grain, they are present right the way through development, following pollination, since they are comprised of enzymes and other proteins important in metabolism. Zeins, formerly termed endosperm specific proteins [14], are made up of free a-subunits (FII), b-, d- and bound a-subunits (FIII) and c-subunits (FIV), and contain very low amounts of lysine and tryptophan. Located in the endosperm only and more specifically in protein bodies, they are synthesized for a period after pollination and consequently later than non-zeins [14,27]. Furthermore, a linear correlation has been established between the absolute amounts of zeins and true proteins accumulated in the developing endosperm originating from wild-type and mutant inbred lines [11]. The straight line has a slope corresponding to the relative rate of zein accumulation and is independent of growth conditions of the plant during development of the grain. The x-intercept provides an approximate value of the amount of non-zeins accumulated in the endosperm, prior to that of zeins [11]. Quantitatively, zeins make up 58% and 70% of the total nitrogen present in the mature
550
J. Landry et al. / Plant Physiology and Biochemistry 43 (2005) 549–556
grain and endosperm, respectively, of the commercial maize variety Inra 260 [14]. Such characteristics result in a deficiency in the grain of lysine and tryptophan, and are the cause of its poor nutritional quality, which is not improved by agricultural and breeding practices, since the proportion of nonzeins in protein decreases with increasing protein content. The discovery that the grain of mutants such as opaque-2 [19] and floury-2 [20] contain higher concentration of lysine than their wild-type counterparts (due to a reduction in the amount of zein in the endosperm), has opened the way for seeking other mutants displaying similar traits. The response of the endosperm following such mutations has been estimated by quantifying their zein or lysine content, together with that of crude proteins, the results being expressed on a dry matter or a crude protein basis, or more rarely on a per endosperm basis. These data are to be treated with caution, due to the underestimation of zeins caused by incomplete extraction, or to their overestimation originating from the presence of NPN in alcoholic extracts, and to the fact the level of NPN had not been determined. As a part of collaborative study concerning the influence of a range of opaque mutations on maize lysine metabolism, we have characterized endosperm samples from a biochemical standpoint, by analyzing the nitrogen fractions and by expressing the data on a dry matter basis [1–3]. In the present investigation, the same mutant samples were examined from a physiological standpoint, by determining the weight proportions of various tissues of the grain, together with the concentrations of eight nitrogen fractions expressed on a per endosperm basis. Zein concentrations were correlated with that of true proteins and compared with the corresponding values obtained from six wild-type inbred lines and their opaque-2 (o2) versions and from the floury (opaque) part of endosperms of eight other wild-type inbred lines. These comparisons have allowed a further insight into the physiological regulation of the accumulation of zein proteins in opaque endosperms.
2. Results 2.1. Grain weight and weight proportions of various endosperm tissues The grain weight, together with the weight proportions of endosperm, embryo and pericarp for each of mutant lines examined in this study, are shown in Table 1. It can be seen that there is: 1) a large variability in the grain weight, which can be more than 100% depending on the genotype. It is noteworthy that when compared to 21 wild-type lines grown under close growth conditions, the mutants Oh43o1, B37o7, Oh43fl2 and W22o13 have grain of similar weight, whereas others have lighter grains (data not shown); 2) an independence of the proportion of pericarp in relation to genotype and grain weight; 3) a lower weight proportion of endosperm compensated by a higher proportion of the embryo in the case of the Oh43o2 line; and 4) an independence of weight proportion of the endosperm and embryo for non-o2 genotypes, the proportions being similar to those observed for 21 wild-type lines. 2.2. Distribution of nitrogen fractions To better characterize proteins from the mutants, we have isolated nine nitrogen fractions, namely: free amino acids (FAA), NPN, albumins + globulins, alcohol-soluble zeins (E3), pH 10 buffer-soluble zeins (E4), total zeins, true glutelins, non-zeins (NZ) and total true proteins. Table 2 lists the amounts of these different fractions in the endosperm, together with those of crude protein, and percentage of crude protein in the dry matter. The amounts of the same fractions originating from three wild-type lines Oh43+, W22+ and B37+, used as background for the mutations are also listed in Table 2 for comparison. Comparisons of the fraction amounts in the various genotypes were done by analyses of variance, and multiple comparisons of means using the Bonferroni correction with ␣ = 0.05 (not shown). The following results emerge: 1) the three wild-type lines are similar for salt-soluble nitrogen
Table 1 Grain weight and weight proportions of the principal tissues of the maize grain according to genotype Genotype Oh43o1 Oh43o2 B79o5 B37o7 W22o10 W22o11 W22o13 Oh43fl1 Oh43fl2 (non-o2) a,b 21 lines + a,c a
Wg (mg per grain) 210 121 128 209 125 102 149 135 190 156 ± 42 232 ± 35
Wendosperm/Wg (%) 79.4 70.6 81.2 82.1 83.4 82.2 77.3 83.0 80.8 82.3 ± 1.0 81.8 ± 2.2
Mean ± standard deviation. Opaque and floury mutants other than o2. c Wild-type lines selected for their differences in vitreousness.
b
Wgerm/Wg (%) 13.3 21.3 13.5 10.9 8.7 10.2 15.1 9.9 12.9 11.7 ± 2.3 11.0 ± 1.3
Wpericarp/Wg (%) 7.2 8.0 5.2 7.0 8.0 7.6 7.4 7.0 6.4 7.0 ± 0.9 7.2 ± 1.4
J. Landry et al. / Plant Physiology and Biochemistry 43 (2005) 549–556
551
Table 2 Distribution of nitrogen compounds in the maize endosperm according to genotype (data expressed as mg protein per endosperm) Genotype Oh43o1 Oh43o2 B79o5 B37o7 W22o10 W22o11 W22o13 Oh43fl1 Oh43fl2 Oh43+ B37+ W22+
FAA a 0.16 0.12 0.21 0.22 0.56 0.23 0.14 0.20 0.23 0.13 0.30 0.31
NPN b 0.67 0.36 0.47 0.56 0.89 0.55 0.38 0.39 0.66 0.30 0.50 0.60
E1,2 c 1.78 1.71 1.46 2.21 1.63 1.22 1.62 1.11 2.27 0.63 1.19 1.30
A+Gd 1.11 1.35 0.99 1.65 0.74 0.67 1.24 0.72 1.61 0.33 0.69 0.70
E3 e 18.17 4.56 14.05 16.21 7.84 7.40 10.93 13.72 15.03 12.25 15.07 17.03
E4 f 0.94 0.53 0.74 0.89 0.67 0.55 0.75 0.60 0.71 0.60 0.70 0.80
Zeins g 19.11 5.09 14.79 17.10 8.51 7.95 11.68 14.32 15.74 12.85 15.77 17.83
E5,6 h 6.97 5.46 5.32 7.01 3.87 4.96 5.59 4.49 6.26 3.00 4.35 5.10
NZ i 8.08 6.80 6.31 8.65 4.61 5.62 6.59 521 7.87 3.33 5.04 5.80
REi j 27.85 12.25 21.57 26.31 14.01 14.12 18.65 19.92 24.27 16.48 21.31 24.23
RtP k 27.19 11.89 21.10 25.75 13.12 13.57 18.27 19.53 23.61 16.18 20.81 23.63
P/DM% l 11.6 8.7 14.8 11.1 9.8 12.1 11.1 12.8 11.2 10.8 13.1 13.3
a
FAA: free amino acids (crude TCA extract). NPN: non-protein nitrogen (hydrolyzed TCA extract). c E1,2: salt extract (0.5 NaCl, 4 °C). d A + G: albumins + globulins (E1,2–NPN). e E3: alcohol extract. f E4: pH 10 buffer extract (water-soluble zeins). g Zeins: E3 + E4. h E5,6.: residue: true glutelins. i NZ: non-zeins: true proteins–zeins. j REi: crude proteins. k RtP: true proteins (REi–NPN). l Crude protein content in dry matter (DM). b
(E1,2) and albumins + globulins, but statistically different for E3, E4 and E5,6, 2) compared to wild-type lines, o11, fl1, o10, o13 and o5 have the same amount of salt-soluble nitrogen, while fl2, o1, o2 and probably o7 are richer; 3) o10, fl1, o11 and o5 have an albumin + globulin content similar to that of wild-type lines while fl2, o2, o13, o7 and probably o5 are richer, the highest content being shown for o7; 4) the level of zeins, compared to that of Oh43+, is not different for fl1 and fl2, but it is significantly higher for o1 and lower for o2; it is lower for o13 and for o11 and o10, when compared to that of W22+; 5) regarding the amounts of non-zeins the following hierarchy is highlighted: o1 ≅ fl2 > o2 > fl1 > Oh43+; further, o7 and o13 have higher amounts than their corresponding wild-type lines, while o10 has lower amount and o11 is similar to W22+; 6) regarding amounts of true proteins the hierarchy is: o1 > fl2 > fl1 > Oh43+> o2; further, o7 and o13 are not significantly different from their wild-type counterparts, but o10 and o11 have lower amounts than W22+. On the other hand a slightly different picture is seen when the data are expressed as a percentage of total crude proteins (data not shown). Thus zeins range from 41.5 (o2) to 71.9% (fl2), increasing to 68.6% in o1 only. These percentages are lower than those found for the wild-type lines (74.8% on average). Finally the hierarchy of the magnitude of total crude protein varies according to the basis (per endosperm or dry matter) used for expressing the data. 2.3. Relationship between zeins and true proteins A closer examination of the data presented in Table 2, brings out a parallelism between the quantitative variations of zeins and those of true proteins among the mutants.
To further investigate the effect of the opaque mutation on the accumulation of zein in the endosperm, the amount of zein expressed per endosperm (WZ) has been plotted against the amount of true protein (WtP) for all the nine mutants used in this study. A linear correlation is observed (Fig. 1) for the eight non-o2 mutants, with a regression line for the equation: WZ = –(1.62 ± 1.13) + (0.752 ± 0.054) WtP of r2 = 0.970 (1). On the other hand, the two parallel lines, each having a ver-
Fig. 1. Total zeins (E3 + E4) in whole endosperms of all lines used in the study: opaque maize (.), wild-type inbred lines (*) and their o2 versions (C). The regression line is relative to non-o2 mutants; and the underlined data points to Oh43o2 inbred lines. Data from present study (.) and from Landry et al. [16] (*, C).
552
J. Landry et al. / Plant Physiology and Biochemistry 43 (2005) 549–556
tical distance 2.31 sˆyx = 1.73 mg, constitute the 95% confidence limits. The data point for Oh43o2 is seen to be outside of the lower limit. The regression line related to the zein accumulation in the mature endosperm of six wild-type lines, taken as reference, has the equation: WZ = –(0.062 ± 0.061) + (0.792 ± 0.029) WtP with r2 = 0.987 (2). It is parallel to the above one, as the slopes are not significantly different, and merges practically with the upper 95% confidence limit, which shows the absence of discontinuity between wild-type and mutants, regarding the levels of zeins and true proteins in the endosperm. In contrast, the data points concerning zein accumulation in the endosperm of six o2 versions of these inbred lines, including the Oh43o2 genotype, are scattered in confidence range, which provides evidence that the regression line determined for non-o2 mutants can also be fitted for o2 ones. The discrepancy recorded between the Oh43o2 sample characterized in the present study and that examined previously [15,16] for zein and true protein content, highlights the intragenotype variability in the distribution of nitrogen fractions. To demonstrate the general character of this regression line, we have also included data previously published by Di Fonzo et al. [8]. These authors determined the effect of the o7 mutation on the distribution of six nitrogen fractions (NPN, FI....FV) in the developing endosperm of W22o7. At maturity, the endosperm contained 7.5 mg of true proteins and 3.7 mg of zeins (FII + FIII + FIV). The absence of any significant discrepancy between these experimental values and those deduced from the regression line (WZ = 4 mg for WtP = 7.5 mg), allows six values obtained from the data of Di Fonzo et al. [8] (five of which correspond to immature endosperms), to be introduced into the new regression, which has the following equation: WZ = –(2.21 ± 0.35) + (0.779 ± 0.021) WtP with r2 = 0.980 (3). On the other hand, the two parallel lines, each having vertical distances 2.2 sˆyx = 1.3 mg, delineate a narrower 95% confidence range (Fig. 2). This regression represents the average variations of zein accumulation in developing and mature endosperms of non-o2 mutants. The data points around the wild-types inbred lines are seen to be excluded from the regressions, whereas those around their o2 versions belong to it, confirming the above conclusions. Maize grains with the opaque phenotype have endosperms with a soft (floury) texture only. Some similarities between the floury portion of wild-type endosperms and the o2 endosperm have been reported as for the appearance of starch granules, the limited number and size of protein bodies, and the distribution of nitrogen fractions [17,21]. We have attempted to establish whether these similarities can be revealed by the relationship connecting zein and true protein content, expressed per endosperm. In this context, the amount of zein present in the floury region of endosperms originating from 8 wild-type inbred lines has been plotted against the corresponding true protein content (Fig. 2). The zein content of floury endosperms, the richest in true proteins (three samples), is able to fit the regression (3) defined for non-o2 samples. The data points of the three samples with a medium
Fig. 2. Total zeins (E3 + E4) in whole endosperms of opaque maize (.), wild-type inbred lines (*) and their o2 versions (C), W22o7 inbred line (D) and in the floury portion of mature endosperms from wild-type inbred lines (e). Data points (D) the lowest in zeins, correspond to immature stages. The regression line is relative to non-o2 and W22o7 maize. The data point (.) outside the 95% confidence range, corresponds to Oh43o2. Data from present study (.), from Landry et al. [16] (*, C), from Di Fonzo et al. [8] (D) and from Landry et al. [17] (e).
content of true proteins are located at the upper 95% confidence limit of the regression, the two others being outside. For a high level of true proteins there is a quantitative similarity between the relative contents of zeins in floury portion of endosperm of wild-type lines and in the whole endosperm of mutant lines. 3. Discussion 3.1. Estimation of zein content In the present study, zeins have been considered as the proteins isolated in extracts E3 and E4. E4 is a minor (2.9–4.8% of the total nitrogen) fraction, which is ill-defined when it is subjected to polyacrylamide gel electrophoresis in the presence of SDS. E4 has an amino acid composition intermediary between those of zeins and true glutelins, but the lysine content is closer to that of zein than that of true glutelin [17]. A good agreement was reported between the endosperm content in FII + FIII + FIV (or E3 + E4) determined experimentally and that estimated from the lysine content of the endosperm by assuming a lysine content of zero for zeins and 7% for non-zeins [12]. Furthermore, the (E3 + E4)/E3 ratio calculated from the data of Table 2 for non-o2 mutants, has an average value of 1.06 ± 0.01. Assuming that only half of the proteins present in E4 are zeins, leads to an overestimation of 3%. This is lower, if small traces of zeins are present in the residue (FVI). The protein content of E3 is also given in Table 2 for comparison with other works dealing with the content of alcohol-soluble zeins only (see below). 3.2. Relationship between zeins and true proteins of non-o2 opaque mutant endosperms The linear relationship Wz = a + bWtP connecting zein and true protein content of endosperm, is characterized by two
J. Landry et al. / Plant Physiology and Biochemistry 43 (2005) 549–556
parameters: the slope b and the y-intercept a. The slope b, defined as relative rate of zein accumulation, has been shown to be independent of growth conditions but dependent of genotype [11]. In the present case the slope b is found to be practically independent of genotype, wild-type or non-o2 opaque mutant, revealing a similarity in the relative rate of zein accumulation. Thus, under the growth conditions used for obtaining the samples analyzed in the present study, approximately 0.78 mg is synthesized as zeins, when the wild-type or mutant endosperms accumulate 1 mg of true protein. The y-intercept a, which is negative, allows an approximation of the magnitude of the x-intercept (–a/b). Since b is considered as constant, the absolute value of a is proportional to the x-intercept, thus the value of the latter gives an estimate of the level of non-zeins accumulated prior to zein synthesis. This value may be a slight overestimation, since zein synthesis starts progressively and not abruptly as it is depicted in Fig. 2. Under the growth conditions used in the present study, the endosperms of non-o2 mutants with respect to those of the wild-type, synthesize a comparable or greater amount of non-zeins, before synthesizing zeins at a similar relative rate, the final amount of these proteins being dependent on the genotype. The same process would be also true for the floury portion of wild-type endosperm, when compared with that occurring for horny portion of the flouriest endosperms, in agreement with a strict control of the spatial and temporal expression of the zein genes [7]. The present data bring out a great variability in zein content with the introduction of a given mutant gene in different wild-type backgrounds. This is particularly marked, when W22o7 is compared with B37o7 (see Fig. 2) or Oh 43fl2 (W Z = 15.7 mg for W tP = 23.6 mg) with R807fl2 (WZ = 7.6 mg for WtP = 14.8 mg as calculated from the data of Sodek and Wilson [25]). In other words, a description of the effect of a mutant gene on the accumulation of zeins, without specifying the wild-type background in which it has been introduced, can only be incomplete. Interestingly, the endosperm content of zeins and true proteins of R807fl2 is similar to those found for W22o10 or W22o11. A confirmation of the above statement is provided by analyzing the data reported by Balconi et al. [4]. In a study similar to this, the investigators determined the protein distribution (FI, FII + FIII & FIV + FV + FVI) in developing (two stages) and mature endosperms of nearly isogenic inbred lines of A69y+, o1, o2, o9, o11, fl1 and fl3. Fig. 3 compares the data of Balconi et al. [4] and that reported here, for the amounts of zeins (FII + FIII) and (E3) per endosperm, plotted against the amount of total true proteins. A noteworthy similarity and a marked linear relationship can be seen. The regression line involving all data points including those relative to o2 mutant but not those pertaining to the earliest stage of development (because at this stage the relative rate of zein accumulation is still increasing and has not yet reached its definitive and constant value characterizing the later stages) has for equation: WZ = –(1.72 ± 0.46) + (0.742 ± 0.025) WtP with r2 = 0.982 [4].
553
Fig. 3. Zeins (E3) in mature endosperms isolated from the grains of opaque maize (.) and in the developing and mature endosperms from the wild-type A69y inbred line (*) and the corresponding o2 (C), o1, o9, o11, fl1 (n) and fl3 (M) versions. The regression line is relative to the maize lines originating from A69y. Data points outside the 95% confidence range correspond to A69yo2 and Oh43o2. Data from present study (.) and from Balconi et al. [4] (*, C, n and M).
Several observations can be made when the data of Balconi et al. [4] are compared with the present results: 1) the slopes of regression lines (2) and (4) are not significantly different. The same is true of the relative rate of zein accumulation, suggesting practically identical growth conditions prior to pollination. However, the ratio of these slopes amounts to 1.05 and is similar to the mean of the ratios (E3 + E4)/E3 calculated above (1.06), agrees with the significant discrepancy due to the presence of proteins present in the E4 extract; 2) the data points relative to o2 mutants are located at the lower 95% confidence limit, suggesting a major accumulation of non-zeins prior to that of zeins, or a reduction the rate of zein accumulation; 3) fl3 exhibits the same behavior as the other non-o2 mutants, the low content in zeins paralleling that of the true proteins; 4) the regression equation (4) includes the values related to the wild-type line A69y+, providing further evidence for the lack of discontinuity between wild-type and mutant samples, regarding the amounts of zeins and true proteins expressed per endosperm. In contrast, the lysine content of the crude proteins of A69yo1 (4.2%), as determined by Balconi et al. [4] using a colorimetric method, is inconsistent with that estimated as 1.9% from the zein content of true proteins [12]. This latter value is close or identical to that reported for the crude proteins of W64Ao1 by Mertz [18] (1.7%), and by Hunter et al. [10] (1.9%, by taking into account of conversion factor of 5.7), indicating that A69yo1 cannot be considered as a highlysine genotype. 3.3. Relationship between zeins and true proteins of opaque-2 endosperms The present data, together with those taken from the literature suggest a process of zein accumulation in the o2
554
J. Landry et al. / Plant Physiology and Biochemistry 43 (2005) 549–556
Fig. 4. Zeins (E3) in immature (D, a, M, e) and mature (D, a, M, e, C, ., m, ▼) endosperms isolated from the grains of opaque-2 maize: W64Ao2 (D) from Soave et al. [24], (a) from Wall and Bietz [26], B37o2 (M) from Salamini et al. [22], A69yo2 (e) from Balconi et al. [4], o2 inbred lines (C) including Oh43o2 (underlined) and B37o2 (double underlined) from Landry et al. [15]; Oh43o2 (.) from present study; Inrao2 (m) from Landry and Moureaux [14]; and R802o2 (▼) from Sodek and Wilson [25]. For each of genotypes D, a, M, e, C the mature state corresponds to the data point located the most to the right.
endosperm slightly different from that described above for other mutants. In fact, some data points concerning the o2 mutants are sometimes outside, or near to the lower 95% confidence limit of the regression lines. To better assess the effect of the o2 gene upon zein accumulation, it is necessary to know the relationship between zeins and true proteins in immature o2 endosperms. In this context we have analyzed some data taken from the literature [22,24,26] as depicted in Fig. 4. This analysis brings out two processes of zein accumulation, which leads practically to the same final level of zeins in the endosperm. In the first case [22,26] zeins are synthesized continuously until maturity with relative rates (0.405 for W64Ao2 and 0.490 for B37o2) that are lower than those recorded for wild-type backgrounds (0.803 for W64A+ and 0.611 for B37+), grown under the same conditions. In the second case [24], two phases are observed for the zein accumulation in W64Ao2. The first is characterized by zein synthesis at a high relative rate (0.706), close to that found for non-o2 mutants (0.735), the second by the synthesis of zeins at a very low rate and non-zeins at a high rate. It is noteworthy that the relative rate of 0.706 is higher than that (0.599) found for the wild-type background, grown under different conditions [11] and it is the only example in which the relative accumulation is observed to take place in two phases. The fact that two processes of zein accumulation are seen for the same genotype, reveals the impact of different growth conditions. The cessation of zein synthesis could be related to the failure of protein bodies to store them, which could promote the synthesis of non-zeins by a compensatory effect in some cases, but it could also result from the cessation of protein synthesis, as suggested by the data from Habben et al. [9]. The latter authors reported the same lysine content in W64Ao2 endosperm at mid-maturity as maturity, providing
Fig. 5. Zeins (E3) in immature and mature endosperms isolated from the grains of opaque maize: W64Ao2 (D) from Soave et al. [24], (a) from Wall and Bietz [26]; W22o7 (▼) from Di Fonzo [8]; and A69Yfl3 (m) from Balconi et al. [4]. For each of genotypes the mature state corresponds to the point located the most to the right. Note that the data point relative to maturity for A69Yfl3 (m) corresponds to that relative to W64Ao2 (D) at midmaturity (data point preceding that located the most to the right).
evidence that the non-zeins were not synthesized during the second phase of maturation. In fact, plotting the zein content against the true protein content in the mature endosperms of various o2 genotypes, gives a scatter diagram highlighting a marked effect of genotype and growth conditions on the relative rate of zein accumulation. Thus, the relative rate of zein accumulation would vary from 0.40 to 0.71. A variability of the same amplitude (from 0.43 to 0.80) is seen for this parameter when Illinois Low Protein is compared to Illinois High Protein [11]. It is noteworthy that the data points from Wall and Bietz [26] for W64Ao2, from Salamini et al. [22] for B37o2, from Sodek and Wilson [25] for R802o2 and from Landry [12] for Inrao2 are on the outside of the lower 95% confidence limit, indicating an effective reduction in the relative rate of zein accumulation in respect to that observed with the wild-type backgrounds. The actual efficiency of the o2 mutation to reduce zein accumulation may be seen only with some samples, the conditions for obtaining the reduction, being ill defined. This variation could explain the lack of precise conclusions about the action of the o2 gene upon zein accumulation, despite numerous studies. On the other hand, the amount of non-zeins accumulated prior to zein synthesis, ranges from 2.2 and 2.9 mg per endosperm for any mutant line, as deduced from the value of the x-intercept of the regression lines. For eight wild-type lines selected among 21 ones for their differences in vitreousness (Table 1 and [17]) the amount of non-zeins was found to vary from 0.3 (for the horniest endosperms) to 2.7 mg (for the flouriest endosperms) (Landry, unpublished data). Therefore, a higher level of non-zeins in the endosperm prior to the zein synthesis, is not a characteristic specific to the presence of the opaque gene but to a floury nature. From the foregoing, the conclusions drawn about the accumulation of zeins in the endosperm of non-o2 mutants remain valid in the case of o2 mutation, as can be seen from the data depicted in Fig. 5. Thus, the levels of zeins and true proteins
J. Landry et al. / Plant Physiology and Biochemistry 43 (2005) 549–556
in the mature endosperms of mutant line A69yfl3 are seen to be approximately the same as those found in the W64Ao2 endosperm at mid-maturity by Soave et al. [24], indicating a reduction in metabolism by 50%. In the case of mature endosperm of W22o7 these same levels are identical to those reported by Wall and Bietz [26] for mature endosperm of W64Ao2. However, the o2 mutation would differ from non-o2 opaque mutations, as the amount of zeins accumulated in the mature endosperm is reduced, irrespective of nature of the wild-type background. A similar quantitative reduction of zeins in the mature endosperm is also observed with the introduction of the o7 and fl2 genes, but such an alteration would be limited to some genetic combinations. 3.4. Molecular basis for the opaque mutation To our knowledge, the molecular basis for the opaque mutation is known for o2 and fl2 only. The O2 gene encodes a transcriptional activator that regulates expression of 22 kDa a-zein and 14 kDa b-zein genes as well as other genes not directly implicated in zein synthesis, including enzymes involved in amino acid and carbohydrate biosynthesis [1–3,6,9,10]. Almost all o2 mutants characterized have an altered O2 protein, promoting alteration in zein accumulation and in endosperm metabolism. These large pleiotropic effects may be involved in the particular relationships between zeins and true proteins, as compared to other mutants. The fl2 mutation has originated from a defective signal peptide of a 22 kDa a-zein, thus altering its processing [5]. Many other endosperm mutants have been reported to have opaque phenotype. These includes a series of the so classified opaque and floury mutants. There is no evidence that the genes encoding these phenotypes has any to do with transcription factors. They do alter physical, chemical and metabolic properties of the endosperm. The work described here is an attempt to connect all these mutants to a concerted endosperm developmental and metabolic chart.
555
types grown under various conditions. The same also holds for zeins from high-lysine genotypes. The high-lysine content is related to a reduction of the true protein level, and thereby of zein level. In some cases there would be a relative rate of zein accumulation, lower than those recorded for wildtype inbred lines.
5. Methods 5.1. Plant material The maize (Zea mays L.) genotypes Oh43o1, Oh43o2, B79o5, B37o7, W22o10, W22o11, W22o13, Oh43fl1 and Oh43fl2 were grown in a glasshouse in the field station of the Genetics Department, Piracicaba, University of São Paulo (Brazil) during 1999–2000 summer season. The wild-type (+) and opaque-2 (o2) versions of the six following inbred lines: W64A, W22, W23, Oh43, F2 and B37, were grown in a glasshouse at Orsay near Paris (France) in 1994 and 1995, (W23+ and o2) were also grown as references. All genotypes but F2 and F2o2 were obtained from the Maize Genetics Corporation Stock Center (University of Illinois, UrbanaChampaign, IL). Eight wild-type inbred lines (COEST6, F113, A188, CO255, LH74, CM105, F7 and ARGL256) selected for their differences in vitreousness, were grown in a glasshouse in Orsay during 2000 and were used for the isolation of the opaque portion of their endosperms. Endosperms, germs and pericarps were isolated by hand dissection of five to eight grains of each line, previously soaked in water for 30 min. Opaque (floury) and horny (vitreous) fractions were isolated from whole endosperm using an adjustable speed grinding wheel [17]. After lyophilization, the fractions were weighed and the dry matter content determined. The endosperms were ground using a ball mill and the dry flour was defatted with hexane, prior to extraction [17]. 5.2. Extraction and quantification of nitrogen compounds
4. Conclusion To conclude, expressing the amount of true proteins on a per endosperm basis is an appropriate measure of the synthetic capacity of the endosperm. Plotting the amount of zeins per endosperm against the amount of true proteins per endosperm, allows the integration of the present data with those already reported in the literature (when they are exploitable) on the same graph and to extract the essential facts concerning the accumulation of zeins in the maize endosperm. The changes in zein amount as a function of true protein, are depicted by: 1) a linear plot in the case of a given genotype, or a set of close genotypes grown under similar conditions. The high values of the coefficient of determination, indicate not a functional relationship but the fact that zeins constitute the major part of true proteins; 2) a set of linear plots forming a scattered plot when these changes pertain to several geno-
Duplicate extracts (E) were obtained by making successive extractions of the endosperm flour with: 1) 0.5 M NaCl, then water (E1,2); 2) water (discarded); 3) 55% (w/w) isopropanol + 0.02% (w/v) dithiothreitol (DTT) (E3); 4) 0.5 M NaCl + 0.02% DTT buffered at pH 10 with sodium borate and sodium hydroxide (E4). The remaining residue (E5,6) contained proteins that could be extracted by SDS (E5) plus insoluble proteins (E6). NPN (E0 by extension) was isolated from precipitated proteins by adding trichloroacetic acid (TCA) to E1,2 extracts, to a final concentration of 10% (w/v). No significant differences have been found between E0 extracted with TCA directly or isolated after protein precipitation [15]. NPN and protein were quantified by the ninhydrin assay of a-NH2 groups (and ammonia) liberated after sample hydrolysis, using an equimolar mixture of 17 amino acids and ammonium sulfate (Pierce) as a standard and a conver-
556
J. Landry et al. / Plant Physiology and Biochemistry 43 (2005) 549–556
sion factor of 1.06 µg of protein for 10 nmol of amino acids. FAA were quantified from TCA solutions without preliminary hydrolysis. NPN and proteins from E0, E1,2 and E4 extracts were hydrolyzed in 3 M NaOH for 45 min [13]. Proteins from flour, E3 extract (after removing solvent) and residue (E5,6) were hydrolyzed in constant boiling HCl at 115 °C for 18 h. From the foregoing NPN, albumins plus globulins, zeins and true glutelins corresponded to a-amino nitrogen contained in E0, E1,2 – E0, E3 + E4, E5,6, respectively.
[7]
5.3. Statistical analysis
[11]
The method of least squares was used for determining the equations of regression together with the 95% confidence range from the standard error of estimate and the number of degrees of liberty. Comparisons of fraction amounts were done using oneway analyses of variance with the genotype as the factor. When a significant F value was obtained, multiple comparisons of means were done with the Bonferroni correction, with ␣ = 0.05.
[8]
[9]
[10]
[12] [13]
[14]
[15]
[16]
Acknowledgements The authors would like to thank Professor Peter Lea, Lancaster University, UK, for reviewing the manuscript and helpful suggestions.
[17]
[18]
[19]
References [1]
[2]
[3]
[4]
[5]
[6]
R.A. Azevedo, C. Damerval, J. Landry, P.J. Lea, C.M. Bellato, L.W. Meinhardt, M. Le Guilloux, S. Delhaye, A.A. Toro, S.A. Gaziola, B.D.A. Berdejo, Regulation of maize lysine metabolism and endosperm protein synthesis by opaque and floury mutations, Eur. J. Biochem. 270 (2003) 4898–4908. R.A. Azevedo, C. Damerval, P.J. Lea, J. Landry, C.M. Bellato, L.W. Meinhardt, M. Le Guilloux, S. Delhaye, A.A. Toro, S.A. Gaziola, V.A. Varisi, P.L. Gratão, Genetic control of lysine metabolism in maize endosperm mutant, Funct. Plant Biol. 31 (2004) 339–348. R.A. Azevedo, P.J. Lea, C. Damerval, J. Landry, C.M. Bellato, L.W. Meinhardt, M. Le Guilloux, S. Delhaye, V.A. Varisi, S.A. Gaziola, P.L. Gratão, A.A. Toro, Regulation of lysine metabolism and endosperm protein synthesis by the Opaque-5 and Opaque7 mutation, J. Agric. Food Chem. 52 (2004) 4865–4871. C. Balconi, N. Berardo, A. Reali, M. Motto, Variation in protein fractions and nitrogen metabolism of developing normal and opaque endosperm mutants of maize, Maydica 43 (1998) 195–203. C.E. Coleman, M.A. Lopes, J.W. Gillikin, R.S. Boston, B.A. Larkins, A defective signal peptide in the maize high-lysine mutant floury-2, Proc. Natl. Acad. Sci. USA 92 (1995) 6828–6831. C. Damerval, M. Le Guilloux, Characterization of novel proteins affected by the o2 mutation and expressed during endosperm development, Mol. Gen. Genet. 257 (1998) 354–361.
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
S. Facio Dolfini, M. Landoni, C. Tonelli, L. Bernard, A. Viotti, Spatial regulation in the expression of structural and regulatory storageprotein genes in Zea mays endosperm, Dev. Genet. 13 (1992) 264– 276. N. Di Fonzo, E. Gentinetta, F. Salamini, Action of the opaque-7 mutation on the accumulation of storage products in maize endosperm, Plant Sci. Lett. 22 (1979) 77–88. J.E. Habben, A.W. Kirleis, B.A. Larkins, The origin of lysinecontaining proteins in opaque-2 maize endosperm, Plant Mol. Biol. 23 (1993) 825–838. B.G. Hunter, M.K. Beatty, G.W. Singletary, B.R. Hamaker, B.P. Dilkes, B.A. Larkins, R. Jung, Maize opaque endosperm mutations create extensive changes in patterns of gene expression, Plant Cell 14 (2002) 2591–2612. J. Landry, A linear model for quantitating the accumulation of zeins and their fractions (a + d, b and c) in developing endosperm of wild-type and mutant maizes, Plant Sci. 163 (2002) 111–115. J. Landry, Quantitation of zeins from the lysine content of grain, endosperm and glutelins from maize, Maydica 48 (2003) 39–54. J. Landry, S. Delhaye, A simple and rapid procedure for hydrolyzing minute amounts of proteins with alkali, Anal. Biochem. 243 (1996) 191–194. J. Landry, T. Moureaux, Distribution and amino acid composition of protein groups located in different histological parts of maize grain, J. Agric. Food Chem. 28 (1981) 1186–1191. J. Landry, S. Delhaye, C. Damerval, Improved method for isolating and quantitating a-amino nitrogen as nonprotein, true protein, saltsoluble proteins, zeins and true glutelins in maize endosperm, Cereal Chem. 78 (2000) 620–628. J. Landry, S. Delhaye, C. Damerval, Effect of opaque-2 gene on accumulation of protein fractions in maize endosperm, Maydica 47 (2002) 59–66. J. Landry, S. Delhaye, C. Damerval, Protein distribution pattern in floury and vitreous endosperm of maize grains, Cereal Chem. 81 (2004) 153–158. E.T. Mertz, Genetic and biochemical control of grain protein synthesis in normal and high lysine cereals, World Rev. Nutr. Diet. 48 (1986) 222–262. E.T. Mertz, L.S. Bates, O.E. Nelson, Mutant gene that changes protein composition and increases lysine content of maize endosperm, Science 145 (1964) 279–280. O.E. Nelson, E.T. Mertz, L.S. Bates, Second mutant gene affecting the amino acid pattern of maize endosperm proteins, Science 150 (1965) 1469–1470. J.L. Robutti, R.C. Hoseney, C.E. Wassom, Modified opaque-2 endosperm. II. Structure viewed with a scanning electron microscope, Cereal Chem. 51 (1974) 173–180. F. Salamini, N. Di Fonzo, E. Fornasari, E. Gentinetta, R. Regiani, C. Soave, C. Mucronate, Mc, a dominant gene of maize which interacts with opaque-2 to suppress zein synthesis, Theor. Appl. Genet. 65 (1983) 123–128. P.R. Shewry, F. Beaudoin, J. Jenkins, S. Griffiths-Jones, E.N. Mills, Plant protein families and their relationships to food allergy, Biochem. Soc. Trans. 30 (2002) 906–910. C. Soave, F. Pioli, A. Viotti, F. Salamini, P.G. Righetti, Synthesis and heterogeneity of endosperm proteins in normal and opaque-2 maizes, Maydica 28 (1975) 83–94. L. Sodek, C.M. Wilson, Amino acid composition of proteins isolated from normal, opaque-2 and floury-2 corn endosperm by a modified Osborne procedure, J. Agric. Food Chem. 19 (1971) 1144–1150. J.S. Wall, J.A. Bietz, Differences in corn endosperm proteins in developing seeds of normal and opaque-2 corn, Cereal Chem. 64 (1987) 275–280. C.M. Wilson, Proteins of the kernel, in: S.A. Watson, P.E. Ramsted (Eds.), Corn: Chemistry and Technology, American Association of Cereal Chemists, St Paul, MN, 1987, pp. 273–310.