Significant progress has been made in elucidating the mechanism of abscisic acid (ABA)-regulated gene expression, including the characterization of an ABA-responsive element (ABRE), which is regulated by basic domain/Leu zipper transcription factors. In addition to the ABRE, a coupling element (CE1) has been demonstrated to be involved in ABAinduced expression. However, a trans factor that interacts with CE1 has yet to be characterized. We report the isolation of a seed-specific maize ABI4 homolog and demonstrate, using a PCR-based in vitro selection procedure, that the maize ABI4 protein binds to the CE-1 like sequence CACCG. Using electrophoretic mobility shift assays, we demonstrate that recombinant ZmABI4 protein binds to the CE1 element in a number of ABA-related genes. ZmABI4 also binds to the promoter of the sugar-responsive ADH1 gene, demonstrating the ability of this protein to regulate both ABA-and sugar-regulated pathways. ZmABI4 complements Arabidopsis ABI4 function, because abi4 mutant plants transformed with the ZmABI4 gene have an ABA-and sugar-sensitive phenotype. Identification of the maize ABI4 ortholog and the demonstration of its binding to a known ABA response element provide a link between ABA-mediated kernel development and the regulation of ABA response genes.

CesA genes are believed to encode the catalytic subunit of cellulose synthase. Identification of nine distinct CesA cDNAs from maize (Zea mays) has allowed us to initiate comparative studies with homologs from Arabidopsis and other plant species. Mapping studies show that closely related CesA genes are not clustered but are found at different chromosomal locations in both Arabidopsis and maize. Furthermore, sequence comparisons among the CesA-deduced proteins show that these cluster in groups wherein orthologs are often more similar than paralogs, indicating that different subclasses evolved prior to the divergence of the monocot and dicot lineages. Studies using reverse transcriptase polymerase chain reaction with gene-specific primers for six of the nine maize genes indicate that all genes are expressed to at least some level in all of the organs examined. However, when expression patterns for a few selected genes from maize and Arabidopsis were analyzed in more detail, they were found to be expressed in unique cell types engaged in either primary or secondary wall synthesis. These studies also indicate that amino acid sequence comparisons, at least in some cases, may have value for prediction of such patterns of gene expression. Such analyses begin to provide insights useful for future genetic engineering of cellulose deposition, in that identification of close orthologs across species may prove useful for prediction of patterns of gene expression and may also aid in prediction of mutant combinations that may be necessary to generate severe phenotypes.Evidence is accumulating to support the notion that some, if not all, of the members of the family of CesA genes in plants encode a glycosyltranferase that plays a key role in the process of cellulose synthesis (for recent reviews, see Brown et al., 1997; Kawagoe and Delmer, 1997, 1998; Delmer, 1999). The deduced proteins from members of this gene family are characterized by the presence of domains that share significant sequence homology with other family 2 glycosyltransferases that are characterized by having conserved motifs surrounding three conserved D residues and a QXXRW motif downstream of D 3 (Campbell et al., 1997). Recent crystallographic evidence supports a model in which the three D residues, in conjunction with a divalent cation, are involved in binding of the UDP-sugar substrate and in catalysis of glycosyltransfer (Charnock and Davies, 1999). In the deduced proteins encoded by most family 2 glycosyltransferases, the domains containing these conserved D residues are consecutive, but the predicted proteins in plants contain a plant-specific conserved and a hypervariable (HVR-2) domain that separate the domains containing these conserved residues. A conserved, extended N-terminal region containing two zinc fingers resembling LIM/Ring domains (Kawagoe and Delmer, 1997) followed by the HVR-1 region also characterizes the plant CesA proteins. Many of these glycosyltransferases, including the plant and bacterial CesA proteins, are predicted to be ancho...

Restriction fragment length polymorphisms have become powerful tools for genetic investigations in plant species. They allow a much greater degree of genome saturation with neutral markers than has been possible with isozymes or morphological loci. A previous investigation employed isozymes as genetic markers to infer the location of genetic factors influencing the expression of quantitative traits in the maize population: (CO159×Tx303)F2. This investigation was conducted to examine the inferences that might be derived using a highly saturated map of RFLP markers and isozymes to detect quantitative trait loci (QTLs) in the same maize F2 population. Marker loci that were associated with QTL effects in this investigation generally corresponded well with previous information where such comparisons were possible. Additionally, a number of previously unmarked genomic regions were found to contain factors with large effects on some plant traits. Availability of numerous marker loci in some genomic regions allowed: more accurate localization of QTLs, resolution of linkage between QTLs affecting the same traits, and determination that some chromsome regions previously found to affect a number of traits are likely to be due to linkage of QTLs affecting different traits. Many of the factors that affected plant height quantitatively in this investigation were found to map to regions also including known sites of major genes influencing plant height. Although the data are not conclusive, they suggest that some of the identified QTLs may be allelic to known major genes affecting plant height.

Retinoblastoma (RB-1) is a tumor suppressor gene that encodes a 105-kDa nuclear phosphoprotein. To date, RB genes have been isolated only from metazoans. We have isolated a cDNA from maize endosperm whose predicted protein product (ZmRb) shows homology to the "pocket" A and B domains of the Rb protein family. We found ZmRb behaves as a pocket protein based on its ability to specifically interact with oncoproteins encoded by DNA tumor viruses (E7, T-Ag, E1A). ZmRb can interact in vitro and in vivo with the replication-associated protein, RepA, encoded by the wheat dwarf virus. The maize Rb-related protein undergoes changes in level and phosphorylation state concomitant with endoreduplication, and it is phosphorylated in vitro by an S-phase kinase from endoreduplicating endosperm cells. Together, our results suggest that ZmRb is a representative of the pocket protein family and may play a role in cell cycle progression. Moreover, certain plant monopartite geminiviruses may operate similarly to mammalian DNA viruses, by targeting and inactivating the retinoblastoma protein, which otherwise induces G1 arrest.Retinoblastoma (RB-1) is a tumor suppressor gene that encodes a nuclear phosphoprotein with a molecular mass of about 105 kDa (1, 2). Inactivation of the RB-1 gene contributes to both familial and sporadic forms of cancer (3). Retinoblastoma and its related p107 and p130 proteins are among the negative regulators of the cell cycle (4, 5). While hypophosphorylated, the Rb protein exerts a growth suppressive effect and arrests cells in G1 phase.Hyperphosphorylation of Rb, or its interaction with viral oncoproteins, prevents Rb from performing its normal functions at G1 phase and enables cells to begin DNA synthesis (6).

Genetic linkage maps were constructed for both maize and tomato, utilizing restriction fragment length polymorphisms (RFLPs) as the source of genetic markers. In order to detect these RFLPs, unique DNA sequence clones were prepared from either maize or tomato tissue and hybridized to Southern blots containing restriction enzyme-digested genomic DNA from different homozygous lines. A subsequent comparison of the RFLP inheritance patterns in F2 populations from tomato and maize permitted arrangement of the loci detected by these clones into genetic linkage groups for both species.

Gibberellins (GAs) are phytohormones required for normal growth and development in higher plants. The Dwarf3 (03) gene of maize encodes an early step in the GA biosynthesis pathway. We transposon-tagged the 0 3 gene using Robertson's Mutator (Mu) and showed that the mutant allele d3-2::Mu8 is linked to a Mu8 element. The DNA flanking the Mu8 element was cloned and shown to be linked to the d3 locus by mapping in a high-resolution population developed by selecting for recombination between d3 and linked genetic marken. To establish unambiguously the identity of the cloned gene as 03, a second mutant allele of 0 3 (d3-4) was also cloned and characterized using the d3-2::MuB sequences as a probe. d3-4 was found to have a nove1 insertion element, named Sleepy, inserted into an exon. A third mutant allele, d3-1, which has the same size 3' restriction fragments as d3-4 but different 5' restriction fragments, was found to contain a Sleepy insertion at the same position as d3-4. On the basis of the pedigree, Sleepy insertion, and restriction map, d3-1 appears to represent a recombinational derivative of d3-4. The 0 3 gene encodes a predicted protein with significant sequence similarity to cytochrome P450 enzymes. Analysis of D3 mRNA showed that the 03 transcript is expressed in roots, developing leaves, the vegetative meristem, and suspension culture cells. We detected reduced 0 3 mRNA levels in the mutant allele d3-5.

Invertase activity is thought to play a regulatory role during early kernel development by converting sucrose originating from source leaves into hexoses to support cell division in the endosperm and embryo. Invertases are regulated at the posttranslational level by small protein inhibitors, INVINHs. We found that in maize (Zea mays), an invertase inhibitor homolog (ZM-INVINH1) is expressed early in kernel development, between 4 and 7 d after pollination. Invertase activity is reduced in vitro in the presence of recombinant ZM-INVINH1, and inhibition is attenuated by pre-incubation with sucrose. The presence of a putative signal peptide, fractionation experiments, and ZM-INVINH1::green fluorescent protein fusion experiments indicate that the protein is exported to the apoplast. Moreover, association of ZM-INVINH1 with the glycoprotein fraction by concanavalin A chromatogaphy suggests that ZM-INVINH1 interacts with an apoplastic invertase during early kernel development. ZM-INVINH1 was localized to the embryo surrounding region by in situ analysis, suggesting that this region forms a boundary, compartmentalizing apoplast invertase activity to allow different embryo and endosperm developmental rates.Kernel development in maize (Zea mays) proceeds through a series of tightly regulated, overlapping stages. After double fertilization, during the prestorage phase, two distinct cell types are established: the triploid endosperm and the diploid embryo. Despite clearly different cell fates, the embryo and endosperm both rely upon photosynthate from source leaves transported through the maternal pedicel region of the developing kernel, ending at the terminal phloem cells. The presence of Suc-hydrolyzing enzymes, which produce hexose sugars from Suc, have been identified as critical for the establishment of the prestorage phase of seed development, and Suc hydrolysis is an important component of realizable plant yield (Cheng and Chourney, 1999;Weschke et al., 2003). The "invertase control hypothesis," largely based on work from dicots (Wobus and Weber, 1999), is supported in maize by the presence of a cell wall invertase, INCW2, localized to the basal endosperm transfer layer (Talercio et al., 1999). Mutations in this gene result in miniature kernels (the mn1 mutation) and have a severely reduced endosperm (Cheng et al., 1996;Vilhar et al., 2002). Recently, the association of a IVR2, a soluble invertase expressed during early kernel development, with seed yield under conditions of limiting photosynthesis suggests that soluble invertases also play a significant role in providing hexose sugars to support cell division during the prestorage phase (Andersen et al., 2002), as has been previously suggested (Zinselmeier et al., 1999).Invertases exhibit complex regulation at the transcriptional and posttranscriptional levels in response to developmental, environmental, and carbohydrate signals (Sturm, 1999). In addition, small (Ͻ20-kD) inhibitor proteins (INVINH) have been associated with invertase preparations in a number of dico...

SummaryA transgenic gene-silencing approach was used to modulate the levels of ethylene biosynthesis in maize (Zea mays L.) and determine its effect on grain yield under drought stress in a comprehensive set of field trials. Commercially relevant transgenic events were created with down-regulated ACC synthases (ACSs), enzymes that catalyse the rate-limiting step in ethylene biosynthesis. These events had ethylene emission levels reduced approximately 50% compared with nontransgenic nulls. Multiple, independent transgenic hybrids and controls were tested in field trials at managed drought-stress and rain-fed locations throughout the US. Analysis of yield data indicated that transgenic events had significantly increased grain yield over the null comparators, with the best event having a 0.58 Mg/ha (9.3 bushel/acre) increase after a flowering period drought stress. A (genotype 9 transgene) 9 environment interaction existed among the events, highlighting the need to better understand the context in which the downregulation of ACSs functions in maize. Analysis of secondary traits showed that there was a consistent decrease in the anthesis-silking interval and a concomitant increase in kernel number/ ear in transgene-positive events versus nulls. Selected events were also field tested under a lownitrogen treatment, and the best event was found to have a significant 0.44 Mg/ha (7.1 bushel/ acre) yield increase. This set of extensive field evaluations demonstrated that down-regulating the ethylene biosynthetic pathway can improve the grain yield of maize under abiotic stress conditions.