The maize endosperm-specific gene shrunken2 (Sh2) MATERIALS AND METHODSIsolation of Sh2 Isoalleles from sh2-ml. The inbred stock containing sh2-ml, the closely linked al-m3, and the activating element, Ac was described (5). Plants were self-pollinated and plump or nonshrunken seed were collected. Remnant mutant seed from ears of corn containing at least one plump seed were saved for planting in subsequent generations. Most revertant Abbreviations: AGP, adenosine diphosphoglucose pyrophosphorylase; 3-PGA, 3-phosphoglyceric acid. tPresent address:

The allosteric enzyme ADP-Glc pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glc, a rate-limiting step in starch synthesis. Plant AGPases are heterotetramers, most of which are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate. The objectives of these studies were to test a hypothesis concerning the relative roles of the two subunits and to identify regions in the subunits important in allosteric regulation. We exploited an Escherichia coli expression system and mosaic AGPases composed of potato (Solanum tuberosum) tuber and maize (Zea mays) endosperm subunit fragments to pursue this objective. Whereas potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes. Potato tuber and maize endosperm AGPases exhibit radically different allosteric properties. Hence, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation. The data herein conclusively show that both subunits are involved in the allosteric regulation of AGPase. Alterations in the small subunit condition drastically different allosteric properties. In addition, extent of 3-PGA activation and extent of 3-PGA affinity were found to be separate entities, mapping to different regions in both subunits.Starch has many applications in industry as well as serving as an energy source in animal and human nutrition. ADP-Glc pyrophosphorylase (AGPase) catalyzes a rate-limiting step in starch synthesis. Modification of the regulatory properties of this enzyme increases starch yields in potato (Solanum tuberosum) tubers, and maize (Zea mays), wheat (Triticum aestivum), and rice (Oryza sativa) seeds (Stark et al., 1992;Giroux et al., 1996;Smidansky et al., 2002Smidansky et al., , 2003. Seed yield and plant biomass increases are conferred by deregulation of endosperm AGPase. Accordingly, AGPase has attracted wide interest for potential crop improvements.Plant AGPases are tissue specific. They are heterotetramers of two small and two large subunits. Though forms of each subunit share significant sequence homology, the small subunit is generally more conserved than is the large subunit (Smith-White and Preiss, 1992). Sequence comparisons of the large subunits divide AGPases into four groups: stem/ tuber, leaf, fruit/root, and endosperm AGPases. Further differences among AGPases are discernable in regulatory properties. While most plant AGPases are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate (Ghosh and Preiss, 1966;Dickinson and Preiss, 1969;Sowokinos and Preiss, 1982;Kleczkowski et al., 1993aKleczkowski et al., , 1993bSikka et al., 2001), extent of regulation varies from organ to organ. Leaf, potato tuber, and tomato (Lycopersicon esculentum) fruit AGPases are sensitive to 3-PGA and phosphate, while endosperm AGPases, except for that of rice (Sikka et al., 2001), are less sensitive to the regulators. AGPases also differ in subcell...

The maize (Zea mays) shrunken-2 (Sh2) gene encodes the large subunit of the rate-limiting starch biosynthetic enzyme, ADPglucose pyrophosphorylase. Expression of a transgenic form of the enzyme with enhanced heat stability and reduced phosphate inhibition increased maize yield up to 64%. The extent of the yield increase is dependent on temperatures during the first 4 d post pollination, and yield is increased if average daily high temperatures exceed 33°C. As found in wheat (Triticum aestivum) and rice (Oryza sativa), this transgene increases maize yield by increasing seed number. This result was surprising, since an entire series of historic observations at the whole-plant, enzyme, gene, and physiological levels pointed to Sh2 playing an important role only in the endosperm. Here, we present several lines of evidence that lead to the conclusion that the Sh2 transgene functions in maternal tissue to increase seed number and, in turn, yield. Furthermore, the transgene does not increase ovary number; rather, it increases the probability that a seed will develop. Surprisingly, the number of fully developed seeds is only ;50% of the number of ovaries in wild-type maize. This suggests that increasing the frequency of seed development is a feasible agricultural target, especially under conditions of elevated temperatures.

The rate of protein evolution is generally thought to reflect, at least in part, the proportion of amino acids within the protein that are needed for proper function. In the case of ADP-glucose pyrophosphorylase (AGPase), this premise led to the hypothesis that, because the AGPase small subunit is more conserved compared with the large subunit, a higher proportion of the amino acids of the small subunit are required for enzyme activity compared with the large subunit. Evolutionary analysis indicates that the AGPase small subunit has been subject to more intense purifying selection than the large subunit in the angiosperms. However, random mutagenesis and expression of the maize (Zea mays) endosperm AGPase in bacteria show that the two AGPase subunits are equally predisposed to enzyme activity-altering amino acid changes when expressed in one environment with a single complementary subunit. As an alternative hypothesis, we suggest that the small subunit exhibits more evolutionary constraints in planta than does the large subunit because it is less tissue specific and thus must form functional enzyme complexes with different large subunits. Independent approaches provide data consistent with this alternative hypothesis.