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AP2 Activity Controls Seed Mass.
AP2 was identified originally as a homeotic gene required for the specification of floral organ identity and was shown to encode
an AP2/EREBP transcription factor (24, 25). Here, we show that loss-of-function ap2 mutants produce large seeds. Our studies using an allelic series of mutants provide strong support that AP2 functions directly in controlling seed mass (Table 2). A recent study provided additional support for this conclusion by showing that transgene suppression of AP2 activity in Arabidopsis produced defective flowers and large seeds (40). Thus, the extent of AP2 gene activity, based on its well defined role in flower development, plays a role in determining seed mass.
ap2 mutations negatively affect plant fertility, primarily through disruption of flower structure that limits the efficiency
of self-pollination in mutant plants. Given that limited availability of resources in the maternal plant appears to account
for the negative correlation between seed number and seed mass (4), ap2 mutants are expected to produce seeds larger than wild-type plants with normal fertility. We confirmed that reduced fertility
did affect seed mass and could account for ≈33% of the increase in average seed weight observed in a strong ap2 mutant (Table 3). However, the remaining, much larger effect of AP2 on seed mass must be attributed to factors unrelated to fertility. The early flowering of plants with strong ap2 mutant alleles (Table 1) provides additional support for the conclusion that the effects of the ap2 mutation on seed mass is not solely due to its effects on fertility. Leaf number is closely related to the availability of
reproductive resources in the maternal plant (13). ap2 mutants produced fewer leaves than wild type and, presumably, have fewer resources available. Yet, ap2 mutants gave rise to significantly larger seeds.
Maternal Effect of AP2 on Seed Mass. Experiments in which wildtype and ap2 mutant genotypes were crossed reciprocally (Table 3) provided an important clue about the mechanisms controlling seed mass by showing that AP2 acts through the maternal genome. Consistent with this finding are analyses of reciprocal crosses in several crop species
showing that seed mass is often influenced by the genotype of the maternal plant (41). One interpretation of our result is that AP2 activity in maternal tissues, potentially including the seed coat, is more important than its activity in the embryo and
endosperm in controlling seed mass. Alternatively, because the maternal plant contributes two genome equivalents to the triploid
endosperm, seed mass may be affected by AP2 gene dosage in the endosperm. A QTL analysis of small- and large-seeded Arabidopsis ecotypes suggests that maternal tissues are important in determining seed mass (13). A number of QTL affecting seed mass colocalized with QTL for maternal traits such as seed number, fruit size, and plant
resources, suggesting that they operate through maternal tissues. The gene identities of these QTL have not been determined,
although AP2 does appear to colocalize to a QTL affecting seed mass at the bottom of chromosome IV (13). Conversely, a recent study used genetic experiments to conclude that AP2 acts through both the endosperm and maternal tissues to affect seed size (40).
The endosperm has been implicated to serve as the site of parent-of-origin effects on seed mass through the imprinting of
genes thought to be involved in enhancing or suppressing endosperm size and, therefore, seed and embryo mass (21–23). Although little is known of the mechanisms mediating parent-of-origin effects on seed mass, no current evidence points
to an involvement of AP2. For example, mutations in three genes, MEA, FIE, and FIS2, each induce endosperm phenotypes that mimic paternal genomic excess (39, 42–44). However, these mutations are inherited differently than AP2 is in that ap2 is a maternal sporophytic mutation, whereas mea, fie, and fis2, are female gametophytic mutations. Thus, AP2 does not act equivalently with these other genes, although they could participate in a common pathway. Two other genes, IKU1 and IKU2, have been proposed to mediate the effects of maternal and paternal dosage on seed size, because mutations in either gene
affect many aspects of endosperm development (45). However, unlike AP2, both iku mutations are sporophytic recessive and not maternal-effect mutations. Thus, the IKU genes are also likely to operate differently than does AP2.
How does AP2 act maternally to control seed mass? Our experiments suggest that AP2 acts, in part, through its effects on both embryo cell number and size (Fig. 2 and Table 4). Others have shown that changes in cell number and size underlie ecotype variations in Arabidopsis seed size (13). Similar to our findings, this study of natural variation concluded that cell number differences are controlled by maternal
factors. However, by contrast to our results with AP2, ecotype differences in cell size were attributed to nonmaternal allelic variation. Our finding that AP2 acts maternally to affect embryo cell size may indicate that it operates through a mechanism that was not uncovered by studies
of natural ecotype variations.
The seed coat has been implicated to influence seed mass. For example, sporophytic mutations in cereals that affect development
of a specialized seed coat tissue responsible for nutrient transfer to the endosperm cause defects in seed development and
size (46–48). ap2 mutants have defective seed coats in that they lack epidermal plateaus and mucilage, their outer integument cells are larger
than those of wild type and are irregular in shape, and mutant seeds are hypersensitive to bleach (24, 49). Although it is not clear whether these defects are directly associated with AP2 effects on seed mass, they emphasize that the maternal component of the seed is defective in ap2 mutants.
AP2, Seed Mass, and Soluble Sugar Metabolism. Our findings that the ap2 mutation acts maternally, potentially through the seed coat, to control seed mass (Table 3) and affects soluble sugar metabolism during seed development (Fig. 4) provide a potential explanation for the increase in embryo cell number in ap2 mutant seeds (Table 4). During seed development, modulation of hexose and sucrose levels has been implicated to control cellular activities (reviewed
in ref. 15). Specifically, a high ratio of hexose to sucrose is correlated strongly with mitotic activity during the early morphogenesis
phase of seed development, whereas a higher proportion of sucrose to hexose is associated with cell expansion and seed filling
during the late maturation phase (14–16). For example, immature fava bean embryos cultured with high concentrations of hexose continue to undergo cell divisions
whereas those cultured with high sucrose concentration enter the maturation phase (15). Thus, cell division during the morphogenesis phase and seed filling during the maturation phase appear to correlate with
changes in hexose and sucrose levels.
ap2 mutations induced changes in the levels of hexose and sucrose in developing seeds. These changes resulted in hexose/sucrose
ratios that reached a higher maximal level in ap2 mutant seeds compared with wild-type seeds and that remained high for a longer period of seed development in ap2 mutants than they did in wild type (Fig. 4). For example, hexose/sucrose ratios remained high at 9 and 11 DAP in ap2 mutants but was very low during the same period of wild-type seed development. We speculate that the altered hexose/sucrose
ratios during ap2 mutant seed development may promote an extended period of cell division that could account for the increase in embryo cell
number observed in ap2 mutants.
AP2 is a transcription factor. Thus, it is likely to affect seed mass by regulating the expression of other genes. Given the
dramatic changes in hexose levels in ap2 mutants, potential targets of AP2 activity are enzymes involved in sugar metabolism, such as cell-wall-bound invertases. Sucrose is transported from photosynthetic
organs to the seed coat, a maternally derived structure, where it is metabolized differently early and late in embryogenesis
(14, 50–52). During the early phase of favabean seed development, cell-wall-bound invertases localized in thin-walled parenchyma, the
innermost seed coat tissue, hydrolyze sucrose. Hexoses formed by the reaction move apoplastically and are transported into
embryo cells. Cell-wall-bound invertase activity decreases during the late phase of seed development, and sucrose is transported
directly into embryo cells where it is hydrolyzed by sucrose synthase. Cell-wall-bound invertase activity has been correlated
with seed cell number both in fava bean cultivars that exhibit variations in seed size and in the maize miniature1 mutant that is defective in the enzyme (15, 53). Thus, AP2 may affect seed mass by controlling cell-wall-bound invertase activity.
We note that two other members of the AP2/EREBP transcription factor family have roles that appear to intersect with those
of AP2. Mutations in the Arabidopsis WRINKLED1 gene lead to defects in carbohydrate metabolism and reductions in seed mass and oil content (38, 54). Arabidopsis AINTEGUMENTA gene is thought to play a role in controlling cell proliferation (55, 56). Ectopic expression of AINTEGUMENTA caused plant organs, including seeds, to increase in size in both transgenic Arabidopsis and tobacco (57). The increase in seed size in transgenic plants appears to result from an increase in cell number during seed development.
Thus, other AP2/EREBP proteins may function in a pathway similar to that of AP2.
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