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a Station de Génétique Végétale, Ferme de Moulon, 91190 Gif/Yvette, France
b Institut National Agronomique Paris-Grignon, 16 rue Claude Bernard, 75231 Paris Cedex, France, and Station de Genétique Végétale, Ferme du Moulon, 91190 Gif/Yvette, France
c Reproduction et Développement des Plantes, ENS-Lyon, 46, allée d'Italie 69364 Lyon cedex 9, France
d Structure et Métabolisme des Plantes, I.B.P., Bât 610, Université Paris-Sud, 91400 Orsay, France
gallais{at}moulon.inra.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Final kernel dry weight is determined by the duration of the grain filling period and by the starch accumulation rate. At maturity, starch represents about 75% of maize kernel weight. Both length of grain filling and starch accumulation are affected by genes from the male parent (Perenzin et al., 1980). Capitanio et al. (1983) observed relationships among grain weight and the endosperm cell number and the rate and duration of grain filling. However, grain weight was not related to starch granule size or three important enzymes involved in starch biosynthesis. Doehlert and Lambert (1991) have compared a hybrid with its two parents for starch metabolism at 20 and 50 d after pollination. At maturity, the hybrid exceeded its parents for endosperm and embryo dry weight. However, they did not observe differences in sucrose and starch amount or in key enzymes of starch metabolism during the grain filling period.
In spite of the importance of the phenomenon of xenia, its physiological and genetic bases are not well understood. The aim of our work on maize was to study its expression and variation during kernel development at the level of fresh and dry weight and of enzyme activities, according to the male and female genotype.
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Materials and methods |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Xenia Effect on Growth During Grain Filling
Two French inbred lines were used : F497 and F252 (Bulant, 1996, Bulant and Gallais, 1998). F497 is an isogenic white kernel line of W64A, a relatively late inbred line with dent kernels; it was used as female. F252 is an early line with a yellow dent kernel; it was used as male. They were grown in fields at Orsay (France) during 1995. Female ears were bagged before silking to avoid uncontrolled fertilization. At anthesis, six plants of F497 were self-fertilized and six others were crossed with F252 as male. Ten kernels were removed from the same ear of the six plants at 14, 21, 28, 39, and 74 DAP to study the time course of kernel fresh, dry weight, and grain moisture. For each sampling, external husks were discarded and a small windows was open on internal husks in the center portion of the ear. Kernels were removed with a scalpel and then husks were replaced in their primitive position and maintained by a small adhesive band. Fresh weight was evaluated immediately; dry weight was evaluated after 48 h in a drying oven at 80°C.
Xenia Effect on Starch Metabolism at 25 DAP
In 1993, in a preliminary study of xenia effect on starch metabolism, activity of ADP-glucose pyrophosphorylase (EC 2.7.7.27, ADPGppase), and glucose, sucrose, and starch contents were studied on endosperm and embryo from selfed and cross-fertilized kernels sampled at 25 DAP from ears of the line F564, selfed and crossed to the line A188. F564 is a flint inbred line and A188 is dent inbred line. Two plants were used in each plot and 15 kernels per plant were sampled. Kernels were rapidly frozen in liquid N2 and stored at -80°C until use. Embryo and endosperm were dissected in ice at 0°C just before starch measurement to preserve enzyme activity.
Xenia Effect on Kernel Weight and Enzyme Activities at 14 and 28 DAP
For this study in 1994, three lines were used: W64A, F564, and F546. F546 is a dent x flint line. Each was selfed and crossed in all possible combinations in a diallel mating design to provide selfed kernels, cross-fertilized kernels, and a comparison of reciprocal crosses. Several sowing dates were chosen to cross lines with different flowering dates. Kernel dry weight was measured from 15-kernel samples taken from the same ear of the plants at 14 and 28 DAP. Twelve plants were sampled in each plot. Kernels were excised from the middle of each ear as described above. Dry weight was evaluated after a 48 h drying at 80°C. To study starch metabolism, six kernel samples were taken from the same 12 plants in each plot. Kernels were rapidly frozen in liquid N2 and stored at -80°C until use. Embryo and endosperm were not separated.
Starch Metabolism Measurements
Activities of ADPGppase, acid invertase (EC 3.2.1.26), neutral invertase (EC 3.2.1.26), sucrose-synthase (EC 2.4.1.13), and glucose, sucrose, and starch contents were measured spectrophotometrically on three kernels pooled per plant and per stage. The samples were weighed and a 4-fold equivalent in volume of extraction buffer was added (50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES]-NaOH [pH = 7.5], 5 mM MgCl2, 2 mM dithiothreitol, 1 mM Na2 EDTA, 200 µL L-1 Triton X100, 6 g kg-1 bovine serum albumin). For the embryo starch metabolism measurement, a 10-fold equivalent in volume extraction buffer was used. The extracts obtained after grinding thawed kernels in 4°C buffer were centrifuged for 10 min in a microcentrifuge at 12000 g and 4°C. All measurements were assayed as described by Pelleschi et al. (1997). ADPGppase activity, and glucose, fructose, and sucrose contents were measured in the supernatant. Activities of acid invertase, neutral invertase, and sucrose synthase (in the sucrose degradation direction) were measured in desalted extracts. Starch was determined after amyloglucosidase (EC 3.2.1.3) treatment from the remaining pellet as described by Prioul et al. (1990). Enzyme activities are expressed per gram fresh weight and per kernel.
Statistical Analyses
For all traits, we analyzed kernels from self fertilization and cross fertilization. From these measurements, we deduced a cross-fertilization effect (R) for each trait as the relative increase of cross-fertilized kernels compared with self-fertilized kernels. Comparison of means was performed with the Student-Newman-Keuls test. In the 3-by-3 diallel experiment, it was not possible to apply complete diallel analysis (Griffing, 1956) with the test of specific effects due to lack of degrees of freedom. Then, a factorial analysis with a female, male, and female x male interaction was applied, including the diagonal, i.e., the self-fertilization cells. Correlations between enzyme activities and carbohydrate contents were computed for absolute and relative cross-fertilization effect. The relative cross-fertilization effect at the level of the whole diallel experiment was computed from the average of the difference between cross and self-fertilized kernels divided by the average of self-fertilized kernels. A test of difference to zero was made through the test of the average of the difference between cross and self-fertilized kernels.
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Results |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Neutral Invertase Activity
At 14 DAP, kernels from cross fertilization had a greater neutral invertase activity than in kernels from self fertilization (+24.1% on average) (Table 4). Neither male effect nor female x male interaction were significant for this enzyme. This means that the male effect was a general effect. By 28 DAP, neutral invertase had decreased by 58%, and there was no difference between cross-fertilized and self-fertilized kernels on average. Furthermore, none of the effects (female, male or female x male) were significant. With the pooled analysis for both sampling dates, male effect was not significant, but female x male interaction was significant (Table 5).
Sucrose Synthase Activity
At 14 DAP, cross-fertilization advantage was 13.3% (Table 4). Neither male effect nor female x male interaction was significant (Table 5). At 28 DAP, sucrose synthase activity had increased by 29%, and there was no difference between cross and self-fertilized kernels and again, neither male effect nor female x male interaction was significant. However, a pooled analysis of the two dates showed a significant female x male interaction. This result suggests that lack of significance for each date was due to a large sampling error. In this pooled analysis, cross-fertilization advantage was significant with the female F564 and for the cross F546 x F564.
Acid Invertase Activity
At 14 DAP, on average, there was no cross-fertilization advantage. There was a significant male effect but no female x male interaction (Table 5). Male effects were quite different from female effects (Table 6)
. F546 showed a negative male effect, whereas the female effect was positive. By 28 DAP, acid invertase activity had decreased by 88%. On average, cross-fertilization effect was negative (-14.6%). There was neither male effect nor female x male interaction. The pooled analysis showed a significant male effect and a male x stage interaction. It was only for this trait that this type of interaction was significant.
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In the diallel experiment, at 14 DAP, on average, there was no cross-fertilization advantage. Glucose and sucrose content strongly depended on maternal genotype. F564 taken as female had the higher carbohydrate content. Male effect was significant, but the female x male interaction was not (Table 5). There was no correspondence between male effect and female effects (Table 6). At 28 DAP, there was also, on average, no cross-fertilization effect. For glucose content, only female effect was significant (Table 5). For sucrose content, the male effect was significant and the female x male interaction was not significant. Again, there was no correspondence between male and female effects (Table 6). The pooled analysis showed a significant male effect for the two carbohydrate contents but was small in comparison to the female effect. There was also a significant female x stage interaction.
Starch Content
In the preliminary experiment at 25 DAP, the endosperm from F546 x A188 had a significantly greater starch content (R = 21%) than F564 selfed (Fig. 1).
In the diallel experiment, at 14 DAP, on average, the cross-fertilization advantage was the greatest value among all the traits studied: +28.8%. This advantage was 77% for kernels from the female F564 (Table 4). The male effect was significant, but the female x male interaction was not (Table 5). At 28 DAP, cross-fertilization advantage was reduced on average to 3.3% and, again, neither male effect nor female x male interaction was significant. However, male effect was at the limit of significance. For both stages, there was no correspondence between male and female effects (Table 6).
Relationships between Cross-fertilization Effects
From the measurements of enzymes and products of the starch metabolic chain, it is interesting to test whether a cross-fertilization advantage for an enzymatic activity is correlated to a cross-fertilization advantage for another enzyme activity or at the product level. Table 7
gives only the correlations which were significant either within or between sampling dates. At 14 DAP, there were significant correlations for cross-fertilization effects between sucrose synthase and ADPGppase (r = 0.80*), sucrose synthase and starch (r = 0.85*), and glucose and starch (r = 0.88*). Significant correlations were not the same at 28 DAP. Correlation between cross-fertilization effects for neutral invertase and sucrose synthase was significant (0.91*), whereas the correlation between starch and sucrose synthase became negative (-0.83*) and correlation between dry-weight and neutral invertase was also negative (-0.89*). However, for some enzyme activities, cross-fertilization effects at 28 DAP were related to cross-fertilization effects at 14 DAP. This was the case for neutral invertase and ADPGppase (r = 0.95**), neutral invertase and sucrose synthase (r = 0.80*), and sucrose synthase and ADPGppase (r = 0.85*). Finally, cross-fertilization effects at 28 DAP for dry weight tended to be negatively related to enzyme activities at 14 DAP (r = -0.81* with neutral invertase, -0.89* with ADPGppase, -0.77 [significant at 0.10] with sucrose synthase). The results were approximately the same when expressing enzyme activities at the kernel level (data not shown).
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Discussion |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Of the enzymes studied, ADPGppase showed the greatest cross-fertilization advantage at 14 DAP (+24.8% on average of all crosses). This advantage was not observed at 28 DAP. The absence of differences between cross and self-fertilized kernels, could be due to the reduction in ADPGppase activity which peaks between 20 and 30 DAP (Prioul et al., 1990, 1994). For our experiment being performed at a given time after pollination, we did not know the position for the maximum enzyme activity of each kernel genotype, which could be different for each. Cross-fertilized kernels which appear to have a more active metabolism, could have an earlier and higher increase in ADPGppase activity and then, an earlier decrease in activity. This hypothesis would explain why kernels from cross fertilization could have a lower ADPGppase activity at 28 DAP than kernels from self fertilization.
Sucrose synthase activity reaches a maximum near 25 DAP (Ou-Lee and Setter, 1985, Prioul et al., 1990); thereafter, it decreases more slowly than does ADPGppase activity. As for ADPGppase activity, the advantage of cross-fertilized kernels at 14 DAP (+13.3%) could be due to an earlier and higher increase in activity. Maximum activity for cross-fertilized kernels would probably be earlier than for self-fertilized kernels. Then, assuming that minimum activity is about the same, this could explain the absence of difference between the two types of kernels at 28 DAP.
As for the sucrose synthase and ADPGppase, the time course of activity for acid and neutral invertase could explain observed results. Acid invertase activity has a maximum at about 15 DAP, then decreases (Ou-Lee and Setter, 1985). Neutral invertase activity is maximal between 8 and 14 DAP, then decreases (Doehlert and Felker, 1987). At 14 DAP, the advantage of cross-fertilized kernels was high (+24.1%) for neutral invertase activity whereas it was not significant for acid invertase activity. At 28 DAP, self-fertilized kernels were similar to cross-fertilized for neutral invertase activity, but were significantly lower for acid invertase activity. This could be interpreted in term of time course activity. In both situations, cross-fertilized kernels were more active than self-fertilized kernels. However, for neutral invertase activity, the maximum was at 14 DAP or before, and then the minimum value was approached for both types of kernels before 28 DAP. For acid invertase activity, the maximum could be after 14 DAP, for example, at 20 DAP, leading to the absence of difference between the two types of kernels at 14 DAP, and then the minimum value was not reached at 28 DAP for self-fertilized kernels, assuming that the maximum activity for cross-fertilized kernels is earlier than for self-fertilized.
For carbohydrate content (glucose and sucrose), at 14 DAP, cross and self-fertilized kernels had on average the same values. Then, it could be concluded that carbohydrate content is mainly controlled by the female. However, the male effect was significant, positive, or negative according to the genotype. At 28 DAP, the situation was nearly similar with a lower carbohydrate content (-6.3% for sucrose) for cross-fertilized kernels. Carbohydrate content could be higher in self-fertilized kernels because their sucrose synthase activity at 14 DAP was lower than in cross-fertilized kernels.
For starch content, in percentage of fresh weight, the advantage of cross fertilization was high (+28.8%) at 14 DAP. Since the maximum peak of ADPGppase activity precedes the maximum starch accumulation rate (Prioul et al., 1994), this could be the result of a greater and earlier ADPGppase activity in cross-fertilized kernels. Unfortunately, there was no measurement of ADPGppase activity before 14 DAP. However, the close correlation between ADPGppase activity measured at 14 DAP and starch content measured at 28 DAP (0.76*) supports such an assumption. At 28 DAP, the cross-fertilization advantage was reduced to 3.3%. However, on a kernel basis, the cross-fertilization advantage for starch quantity was 7.8%. This is the result of a more efficient starch metabolism in cross-fertilized kernels.
Cross fertilization advantage for enzyme activity was expressed on a fresh weight basis. It may be questioned whether the results will change on a kernel basis. Since fresh weight of cross-fertilized kernels was always greater than that of self-fertilized kernels, the cross fertilization advantage in quantity of active enzyme produced per kernel will be even greater in the first stage when enzyme activity is maximum, because relative cross fertilization advantage for dry or fresh weight is also maximum in the first stage. For example, the advantage for ADPGppase becomes 34% at 14 DAP (data not shown). At the minimum of the enzyme activity, when there was no difference between the two types of kernels, the minimum for enzyme quantity correspond to that for dry weight.
Our results, like those of Wang (1947) and Grozmann et al. (1948), showed that cross-fertilization advantage for kernel weight was maximal early in grain development (+19% at 14 DAP). It also corresponds to the period of maximum enzyme activities. The decrease in significance of the cross-fertilization advantage later in grain filling might be related to the decrease in the enzyme activity. During the grain filling linear phase, differences in dry weight between kernels from cross and self fertilization remained approximately constant. Cross-fertilized kernels always had a weight advantage over self-fertilized kernels, which persisted to maturity. Since final kernel dry weight is correlated to the number of cells and starch granules produced during the cell division phase (Reddy and Daynard, 1983), the advantage of kernels from cross fertilization could be due to an earlier initiation of cell division and sink potential. Combined with our previous results (Bulant and Gallais, 1998), the results related to enzyme activity presented here suggest that paternal genes which are expressed almost immediately after fertilization play an important role in developing the sink potential.
It appears that kernel development, even if it is strongly influenced by the maternal genotype, is partially determined by its own genotype. Several authors have observed the effect of paternal genes on different kernel traits. Effects of allopollen were shown on grain filling duration (Poneleit and Egli, 1983, Tsai and Tsai, 1990) and on transformation of starchy endosperm kernels when sweet endosperm kernels were pollinated with starchy endosperm genotype (Kiesselbach and Leonard, 1932), on oil content in the kernels (Curtis et al., 1956), on N accumulation (Tsai and Tsai, 1990), and on endosperm cells and starch granule numbers (Jones et al., 1996). Our results on starch metabolism show an effect of cross fertilization on enzyme activities. Furthermore, male effects were not related to female effects. This could mean that at the kernel level genes function differently according to their male or female origin. Such difference could be due to the fact, that, at one locus, in the endosperm, there are two doses of the female genes and only one of the male and then interactions among identical alleles may change, at the endosperm level, the expression of the genes from the female. The mother plant could also affect kernel enzyme activity as discussed by Bulant and Gallais (1998) for kernel weight at maturity. An effect of the mother plant, in interaction with genes from the male, could also explain the absence of positive significant correlations between the cross-fertilization advantage for grain weight and cross-fertilization advantage for enzyme activities expressed on a fresh weight basis or by kernel.
From a genetic point of view, the advantage of cross fertilization could be interpreted in terms of complementation between male and female genes for some enzymatic systems, i.e., in term of heterosis. The probability of such a complementation with unrelated male and female is high with partial to complete dominance which could explain the cross-fertilization advantage mainly at the level of the end product of a metabolic chain, such as starch. When no cross-fertilization advantage was observed, this could mean that the genes from the male were the same as for the female, for one or several enzymatic systems, assuming that few genes are involved. However, the observed cross fertilization advantage for enzyme activity could also be an early manifestation of heterosis affecting the whole plant (Asby, 1930, 1936).
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ACKNOWLEDGMENTS |
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Received for publication October 27, 1998.
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TOPABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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U. Weingartner, O. Kaeser, M. Long, and P. Stamp Combining Cytoplasmic Male Sterility and Xenia Increases Grain Yield of Maize Hybrids Crop Sci., November 1, 2002; 42(6): 1848 - 1856. [Abstract] [Full Text] [PDF]
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