SummaryWe have developed a novel hybridization platform that utilizes nuclear male sterility to produce hybrids in maize and other cross‐pollinating crops. A key component of this platform is a process termed Seed Production Technology (SPT). This process incorporates a transgenic SPT maintainer line capable of propagating nontransgenic nuclear male‐sterile lines for use as female parents in hybrid production. The maize SPT maintainer line is a homozygous recessive male sterile transformed with a SPT construct containing (i) a complementary wild‐type male fertility gene to restore fertility, (ii) an α‐amylase gene to disrupt pollination and (iii) a seed colour marker gene. The sporophytic wild‐type allele complements the recessive mutation, enabling the development of pollen grains, all of which carry the recessive allele but with only half carrying the SPT transgenes. Pollen grains with the SPT transgenes exhibit starch depletion resulting from expression of α‐amylase and are unable to germinate. Pollen grains that do not carry the SPT transgenes are nontransgenic and are able to fertilize homozygous mutant plants, resulting in nontransgenic male‐sterile progeny for use as female parents. Because transgenic SPT maintainer seeds express a red fluorescent protein, they can be detected and efficiently separated from seeds that do not contain the SPT transgenes by mechanical colour sorting. The SPT process has the potential to replace current approaches to pollen control in commercial maize hybrid seed production. It also has important applications for other cross‐pollinating crops where it can unlock the potential for greater hybrid productivity through expanding the parental germplasm pool.

We have isolated cDNAs from maize (ZGB1) and Arabidopsis (AGB1) encoding proteins homologous to P subunits of guanine nucleotide-binding protein (G protein (9,10) or the transmission of red and blue light-induced signals (11,12). Furthermore, the cloning ofArabidopsis gene GPAI encoding a G protein a subunit designated GPal and its tomato homologue has provided molecular evidence for the existence of G protein-mediated signaling pathway(s) in plants (13, 14). The reports of G protein involvement in a number of different cellular functions raise the possibility that GPal may be involved in more than one signaling pathway. It is possible that G protein f3 subunit(s) might also play a direct role in signaling in plants.In this report, we describe the cloning of the maize ZGB1 and Arabidopsis AGB1 cDNAs encoding proteins that share >41% identity with animal G protein f3 subunits. § This level of homology is greater than that between a known yeast G protein (3subunit and animal ones (15). ZGB1 and AGB1 may represent an additional type of G protein f subunit that is conserved in flowering plants and expressed in roots, leaves, and flowers. MATERIALS AND METHODSMaize Subtracted Library. A maize tassel cDNA library in pCDNAII (Invitrogen) and a maize ear shoot cDNA library in A-Uni-ZAP (Stratagene) were provided by M. Albertsen and G. Huffman, respectively, of Pioneer Hi-Bred International. Biotinylated RNA was generated in vitro from the ear shoot library by using the Gemini Riboprobe system (Promega) with the manufacturer's protocol modified to include 1 mM biotin-11 rUTP (Enzo Diagnostics, instead of photobiotin rUTP) and an increased rUTP concentration (1 mM) in the transcription reaction. A subtracted tassel cDNA library was prepared by hybridization of the biotinylated RNAs with single-stranded DNA from the tassel library as described (16). The second strand of the nonsubtracted DNA molecules was synthesized with Klenow as described (17). One of the obtained clones was designated pPHP2541.S' Rapid Amplification of cDNA Ends (RACE) for PCR. 5' RACE primer extension was performed by using the 5' RACE system (GIBCO/BRL) with leaf and tassel poly(A)+ RNA and the oligonucleotide 5'-GATATCCACAGCCTA-CAGTTG-3' derived from the sequence of the pPHP2541 cDNA insert. The pPHP2541-derived nested primer 5'-GTATTTGATGAGTTGATGGAC-3' and the provided anchor primer were used for PCR amplification with Taq I polymerase (Perkin-Elmer). A clone containing a 0.6-kb PCR product was named pPHP3573.Library Screening, Subcloning, and Sequence Analysis. A A-Uni-ZAP maize tassel cDNA library was screened for a full-length cDNA by using standard conditions and a labeled 0.9-kb insert from pPHP2541 as a probe. A AYES cDNA library from Arabidopsis thaliana (ref. 18; a gift from J. Mulligan) was screened with 32P-labeled 0.9-kb and 0.6-kb maize cDNAs from pPHP2541 and pPHP5373, respectively. cDNAs were subcloned into the Promega vector pGEM7Zf(+) for sequencing of both strands.Southern and Northern Blot Hybridizations. Genomic DNA isol...

To determine the scope of gene expression controlled by the maize transcription factors C1/R and P, which are responsible for activating flavonoid synthesis, we used GeneCalling, an open-ended, gel-based, mRNA-profiling technology, to analyze cell suspension lines of the maize inbred Black Mexican Sweet (BMS) that harbored estradiol-inducible versions of these factors. BMS cells were transformed with a continually expressed estrogen receptor/maize C1 activator domain fusion gene (ER-C1) and either a fusion of C1 and R (CRC), P, or luciferase genes regulated by a promoter containing four repeats of an estrogen receptor binding site. Increasing amounts of luciferase activity, anthocyanins, and flavan-4-ols were detected in the respective cell lines after the addition of estradiol. The expression of both known and novel genes was detected simultaneously in these BMS lines by profiling the mRNA isolated from replicate samples at 0, 6, and 24 hr after estradiol treatment. Numerous cDNA fragments were identified that showed a twofold or greater difference in abundance at 6 and 24 hr than at 0 hr. The cDNA fragments from the known flavonoid genes, except chalcone isomerase (chi1), were induced in the CRC-expressing line after hormone induction, whereas only the chalcone synthase (c2) and flavanone/dihydroflavonol reductase (a1) genes were induced in the P-expressing line, as was expected. Many novel cDNA fragments were also induced or repressed by lines expressing CRC alone, P alone, or both transcription factors in unique temporal patterns. The temporal differences and the evidence of repression indicate a more diverse set of regulatory controls by CRC or P than originally expected. GeneCalling analysis was successful in detecting members of complex metabolic pathways and uncovering novel genes that were either coincidentally regulated or directly involved in such pathways.

To determine the scope of gene expression controlled by the maize transcription factors C1/R and P, which are responsible for activating flavonoid synthesis, we used GeneCalling, an open-ended, gel-based, mRNA-profiling technology, to analyze cell suspension lines of the maize inbred Black Mexican Sweet (BMS) that harbored estradiol-inducible versions of these factors. BMS cells were transformed with a continually expressed estrogen receptor/maize C1 activator domain fusion gene (ER-C1) and either a fusion of C1 and R (CRC), P, or luciferase genes regulated by a promoter containing four repeats of an estrogen receptor binding site. Increasing amounts of luciferase activity, anthocyanins, and flavan-4-ols were detected in the respective cell lines after the addition of estradiol. The expression of both known and novel genes was detected simultaneously in these BMS lines by profiling the mRNA isolated from replicate samples at 0, 6, and 24 hr after estradiol treatment. Numerous cDNA fragments were identified that showed a twofold or greater difference in abundance at 6 and 24 hr than at 0 hr. The cDNA fragments from the known flavonoid genes, except chalcone isomerase (chi1), were induced in the CRC-expressing line after hormone induction, whereas only the chalcone synthase (c2) and flavanone/dihydroflavonol reductase (a1) genes were induced in the P-expressing line, as was expected. Many novel cDNA fragments were also induced or repressed by lines expressing CRC alone, P alone, or both transcription factors in unique temporal patterns. The temporal differences and the evidence of repression indicate a more diverse set of regulatory controls by CRC or P than originally expected. GeneCalling analysis was successful in detecting members of complex metabolic pathways and uncovering novel genes that were either coincidentally regulated or directly involved in such pathways.