Todd E. Young

Increasing vitamin C content of plants through enhanced ascorbate recycling

Vitamin C (ascorbic acid) is essential to prevent disease associated with connective tissue (e.g., scurvy), improves cardiovascular and immune cell functions, and is used to regenerate α-tocopherol (vitamin E). In contrast to most animals, humans lack the ability to synthesize ascorbic acid as a result of a mutation in the last enzyme required for ascorbate biosynthesis. Vitamin C, therefore, must be obtained from dietary sources and, because it cannot be stored in the body, it must be obtained regularly. Once used, ascorbic acid can be regenerated from its oxidized form in a reaction catalyzed by dehydroascorbate reductase (DHAR). To examine whether overexpression of DHAR in plants would increase the level of ascorbic acid through improved ascorbate recycling, a DHAR cDNA from wheat was isolated and expressed in tobacco and maize, where DHAR expression was increased up to 32- and 100-fold, respectively. The increase in DHAR expression increased foliar and kernel ascorbic acid levels 2- to 4-fold and significantly increased the ascorbate redox state in both tobacco and maize. In addition, the level of glutathione, the reductant used by DHAR, also increased, as did its redox state. These results demonstrate that the vitamin C content of plants can be elevated by increasing expression of the enzyme responsible for recycling ascorbate.

Ethylene-Mediated Programmed Cell Death during Maize Endosperm Development of Wild-Type and shrunken2 Genotypes

We characterized the progression of programmed cell death during maize (Zea mays L.) endosperm development of starchy (Su; wild-type) and shrunken2 (sh2) genotypes and tested the involve ment of ethylene in mediating this process. Histological and viability staining demonstrated that endosperm cell death was initiated earlier and progressed more rapidly in sh2 endosperm compared with Su endosperm. Internucleosomal DNA fragmentation accompanied endosperm cell death and occurred more extensively in sh2 endosperm. 1-Aminocyclopropane-1-carboxylic acid levels peaked approximately 16 d after pollination (dap) in Su endosperm and gradually decreased during subsequent development, whereas two large 1-aminocyclopropane-1-carboxylic acid peaks were observed in sh2 endosperm, the first between 16 and 20 dap and the second at 36 dap. Ethylene levels were elevated in sh2 kernels compared with Su kernels, with an initial peak 20 dap approximately 3-fold higher than in Su kernels and a second peak 36 dap approximately 5-fold higher than that in Su kernels. Ethylene treatment of Su kernels resulted in earlier and more extensive endosperm cell death and DNA fragmentation. Aminoethoxyvinylglycine treatment of sh2 kernels reduced the extent of DNA fragmentation. We conclude that ethylene is involved in triggering programmed cell death in developing maize endosperm and is responsible for the aberrant phenotype of sh2 kernels.

Analysis of programmed cell death in wheat endosperm reveals differences in endosperm development between cereals

Although maize endosperm undergoes programmed cell death during its development, it is not known whether this developmental feature is common to cereals or whether it arose inadvertently from the selection process that resulted in the enlarged endosperm of modern maize. Examination of wheat endosperm during its development revealed that this tissue undergoes a programmed cell death that shares features with the maize program but differs in some aspects of its execution. Cell death initiated and progressed stochastically in wheat endosperm in contrast to maize where cell death initiates within the upper central endosperm and expands outward. After a peak of ethylene production during early development, wheat endosperm DNA underwent internucleosomal fragmentation that was detectable from mid to late development. The developmental onset and progression of DNA degradation was regulated by the level of ethylene production and perception. These observations suggest that programmed cell death of the endosperm and regulation of this program by ethylene is not unique to maize but that differences in the execution of the program appear to exist among cereals.

Regulation of programmed cell death in maize endosperm by abscisic acid.

Cereal endosperm undergoes programmed cell death (PCD) during its development, a process that is controlled, in part, by ethylene. Whether other hormones influence endosperm PCD has not been investigated. Abscisic acid (ABA) plays an essential role during late seed development that enables an embryo to survive desiccation. To examine whether ABA is also involved in regulating the onset of PCD during endosperm development, we have used genetic and biochemical means to disrupt ABA biosynthesis or perception during maize kernel development. The onset and progression of cell death, as determined by viability staining and the appearance of internucleosomal DNA fragmentation, was accelerated in developing endosperm of ABA-insensitive vp1 and ABA-deficient vp9 mutants. Ethylene was synthesized in vp1 and vp9 mutant kernels at levels that were 2–4-fold higher than in wild-type kernels. Moreover, the increase and timing of ethylene production correlated with the premature onset and accelerated progression of internucleosomal fragmentation in these mutants. Treatment of developing wild-type endosperm with fluridone, an inhibitor of ABA biosynthesis, recapitulated the increase in ethylene production and accelerated execution of the PCD program that was observed in the ABA mutant kernels. These data suggest that a balance between ABA and ethylene establishes the appropriate onset and progression of programmed cell death during maize endosperm development.

The regulation of gene expression in transformed maize aleurone and endosperm protoplasts. Analysis of promoter activity, intron enhancement, and mRNA untranslated regions on expression.

Gene expression in the aleurone and endosperm is highly regulated during both seed development and germination. Studies of [alpha]-amylase expression in the aleurone of barley (Hordeum vulgare) have generated the current paradigm for hormonal control of gene expression in germinating cereal grain. Gene expression studies in both the aleurone and endosperm tissues of maize (Zea mays) seed have been hampered because of a lack of an efficient transformation system. We report here the rapid isolation of protoplasts from maize aleurone and endosperm tissue, their transformation using polyethylene glycol or electroporation, and the regulation of gene expression in these cells. Adh1 promoter activity was reduced relative to the 35S promoter in aleurone and endosperm protoplasts compared to Black Mexican Sweet suspension cells in which it was nearly as strong as the 35S promoter. Intron-mediated stimulation of expression was substantially higher in transformed aleurone or endosperm protoplasts than in cell-suspension culture protoplasts, and the data suggest that the effect of an intron may be affected by cell type. To examine cytoplasmic regulation, the 5[prime] and 3[prime] untranslated regions from a barley [alpha]-amylase gene were fused to the firefly luciferase-coding region, and their effect on translation and mRNA stability was examined following the delivery of in vitro synthesized mRNA to aleurone and endosperm protoplasts. The [alpha]-amylase untranslated regions regulated translational efficiency in a tissue-specific manner, increasing translation in aleurone or endosperm protoplasts but not in maize or carrot cell-suspension protoplasts, in animal cells, or in in vitro translation lysates.

Cloning of an alpha-amylase cDNA from aleurone tissue of germinating maize seed.

A number of hydrolases are required for the conversion of stored carbohydrate reserves in cereal grains into metabolizable products during germination. a-Amylase (EC 3.2.1.1) plays a key role in this process by catalyzing the endoglycolytic cleavage of amylose and amylopectin, the principal components of starch granules in the endosperm (Bewley and Black, 1985). Studies of a-amylase characterization and expression in cereals have been conducted mainly in barley, wheat, and rice, with very limited information available for maize (Zea mays L.). Multiple isozymes of this enzyme are synthesized in two tissues, the aleurone and scutellum of the embryo, and can be separated on the basis of their pI. In barley and wheat, the isoforms fall into two distinct groups (low pI [ ~ 5 . 5 ] and high pI [?5.5]); however, in rice and maize, there is only one group of isozymes, between pI 3 and 6 (McGregor et al., 1988; Sanwo and DeMason, 1993). To facilitate a more detailed understanding of gene structure and regulation in barley, wheat, and rice, a number of a-amylase genes have been cloned and characterized, and these studies have revealed that the cereal a-amylases are encoded by multigene families (Baulcombe et al., 1987; Knox et al., 1987; Khursheed and Rogers, 1988; Huang et al., 1990; O’Neill et al., 1990). Furthermore, it has been shown that although some of the isozymes are derived from different genes, others differ as a result of posttranslational modifications (Fincher, 1989; Jones and Jacobsen, 1991; Sticher and Jones, 1992). The function and significance of the multiple isozymes are not known. To further advance our understanding of a-amylase expression in maize, we have isolated cDNAs from maize libraries and report here the cloning of a full-length aamylase cDNA (Table I). Three Xgtll cDNA libraries were constructed from aleurone and scutellar tissues of germinating seeds of the maize inbred OH43. To accomplish this, total RNA was extracted approximately 7 d after inhibition from isolated aleurones of both a dent (wild type) and a shrunken2 isogenic line and from scutella of the dent isoline. Poly(A)+ RNA was isolated from each using the PolyATtract mRNA isolation system (Promega) and was subsequently used to synthesize cDNAs according to the specifications of a cDNA synthesis kit (Boehringer Mannheim).

Induction of RNase and nuclease activity in cultured maize endosperm cells following sucrose starvation

The activity of RNases and nucleases in plants often increases following exposure to many types of stress, including prolonged exposure to dark or phosphate starvation. In cereals, the activity of RNases and nucleases is also regulated developmentally during late seed development. In this study, we investigated the effect that the absence of sugar or phosphate in culture medium has on the activity of RNases and nucleases expressed in maize endosperm suspension cells. Withdrawal of sugar from the culture medium resulted in a substantial increase in RNase and nuclease activities, whereas deprivation of phosphate during the same period of growth had no detectable effect on either of these activities. The increase in RNase activity was limited to the neutral RNases, demonstrating that the effect of sugar starvation is specific to one class of RNase. Elimination of asparagine from the medium resulted in a transient reduction in nuclease but not in RNase activity. These observations suggest that sugar starvation constitutes a stress to which maize endosperm responds, in part, by increasing neutral RNase and nuclease activity.