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To improve pod shatter resistance in the important oilseed crop Brassica napus, the phenotypic diversity of B. napus was tested using 80 B. napus varieties for pod shatter resistance by a random impact test. Among these varieties, R1-1 was identified as resistant, while R2, 8908B was susceptible to shatter. To understand the molecular basis for this phenotypic difference based on the candidate gene approach, B. napus FRUITFULL (FUL) homologs were identified and characterized. Two FUL loci in the A and C genomes of B. napus were identified. In the susceptible variety, both BnaA.FUL and BnaC.FUL were expressed in the same tissues. However, the expression level of BnaC.FUL differed in varieties with different pod shatter resistance. In the most resistant variety, R1-1, only BnaA.FUL was expressed, while BnaC.FUL was silenced. Therefore, the functional divergence and differing expression of BnaX.FUL homeologs may significantly affect phenotypic variation, which is an important consequence of allopolyploid evolution. This expression level divergence may be useful for selecting pod shatter resistant lines through marker-assisted selection in B. napus-breeding programs.

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Molecular analysis of floral induction in Arabidopsis has identified several flowering time genes related to 4 response networks defined by the autonomous, gibberellin, photoperiod, and vernalization pathways. Although grass flowering processes include ancestral functions shared by both mono- and dicots, they have developed their own mechanisms to transmit floral induction signals. Despite its high production capacity and its important role in biofuel production, almost no information is available about the flowering process in sugarcane. We searched the Sugarcane Expressed Sequence Tags database to look for elements of the flowering signaling pathway under photoperiodic control. Sequences showing significant similarity to flowering time genes of other species were clustered, annotated, and analyzed for conserved domains. Multiple alignments comparing the sequences found in the sugarcane database and those from other species were performed and their phylogenetic relationship assessed using the MEGA 4.0 software. Electronic Northerns were run with Cluster and TreeView programs, allowing us to identify putative members of the photoperiod-controlled flowering pathway of sugarcane.

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The transition from vegetative to reproductive growth is a critical event in the life cycle of plants. Previous physiological studies have deduced that hormone-like substances mediate this important transition but the biochemical nature of the putative signaling molecules has remained elusive. Recent molecular and genetic studies of key flowering-time genes offer new approaches to understanding the mechanisms underlying the initiation of flowering.

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The life of a plant unfolds as a series of developmental stages, with each stage defined by changes in meristem identity. In maize, there are several distinct stages: the transition from vegetative growth to flowering, the elaboration of the inflorescence, and the formation of flowers. Progress in understanding meristem identity and function has been made by analyzing maize mutants with defects at each of these stages. Recently cloned genes suggest that, although the molecular mechanisms controlling floral organ identity are conserved in maize and other model species, the control of meristem identity during earlier developmental stages might be less conserved.

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Flowering in plants is a consequence of the transition of the shoot apex from vegetative to reproductive growth in response to environmental and internal signals. The indeterminate1 gene (id1) controls the transition to flowering in maize. We show by cloning the id1 gene that it encodes a protein with zinc finger motifs, suggesting that the id1 gene product functions as a transcriptional regulator of the floral transition. id1 mRNA expression studies and analyses of transposon-induced chimeric plants indicate that id1 acts non-cell-autonomously to regulate the production of a transmissible signal in the leaf that elicits the transformation of the shoot apex to reproductive development. These results provide molecular and genetic data consistent with the florigen hypothesis derived from classical plant physiology studies.

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While a large number of cyclins have been described in animals and yeasts, very limited information is available regarding cyclins in plants. We describe here the isolation of cDNA clones encoding four putative mitotic cyclins from maize. All four cyclins were able to induce maturation of Xenopus oocytes, demonstrating that they can act as mitotic cyclins in this system. Northern analysis showed that all four cyclins were expressed only in actively dividing tissues and organs, with a stronger correlation between expression and mitotic activity than is observed with cdc2. The deduced protein sequences suggest that the four maize cyclins belong to the cyclin A and B families identified from animal and yeast studies but that they cannot be described easily as either A-type or B-type cyclins. However, comparison with previously cloned plant cyclins shows that cyclins in higher plants form three distinct structural groups that have been conserved in both monocotyledonous and dicotyledonous species and that cyclins from all three groups are present within a single plant species.

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We have used an antibody against the functional homolog of the cdc2 kinase of maize to localize the p34cdc2 protein within dividing cells of the root apex and the stomatal complex of leaf epidermis. The microtubule cytoskeletal structure of plant cells was visualized concomitantly with a monoclonal antibody specific for [alpha]-tubulin. We found that the cdc2 protein is localized mainly to the nucleus in plant cells at interphase and early prophase. This finding contrasts markedly with the predominantly cytoplasmic staining obtained using antibody to the PSTAIRE motif, which is common to cdc2 and numerous cdc2-like proteins. In a subpopulation of root cells at early prophase, the p34cdc2 protein is also distributed in a band bisecting the nucleus. Double labeling with the maize p34cdc2Zm antibody and tubulin antibody revealed that this band colocalizes with the preprophase band (PPB) of microtubules, which predicts the future division site. Root cells in which microtubules had been disrupted with oryzalin did not contain this band of p34cdc2 protein, suggesting that formation of the microtubule PPB is necessary for localization of the p34cdc2 kinase to the plane of the PPB. The p34cdc2 protein is also localized to the nucleus and PPB in cells that give rise to the stomatal complex, including those cells preparing for the highly asymmetrical divisions that produce subsidiary cells. Association of the p34cdc2 protein with the PPB suggests that the cdc2 kinase has a role in establishing the division site of plant cells and, therefore, a role in plant morphogenesis.

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We describe the isolation of cDNA clones encoding a p34cdc2 homologue from a higher plant, Zea mays (maize). A full-length cDNA clone, cdc2ZmA, was isolated, sequenced, and shown to complement a cdc28 mutation in Saccharomyces cerevisiae. Comparison of the deduced amino acid sequence of the maize p34cdc2 protein with other homologues showed that it was 64% identical to human p34cdc2 and 63% identical to Schizosaccharomyces pombe and S. cerevisiae p34cdc2 proteins. Studies of expression of the maize cdc2 gene(s) by Northern blot analysis indicated a correlation between the abundance of cdc2 mRNA and the proliferative state of the tissue. Southern blot analysis, as well as isolation of another cDNA clone, cdc2ZmB, which is 96% identical to cdc2ZmA, indicates that maize has multiple cdc2 genes.

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We have studied the resistance of cytosine methylated DNA to digestion by the restriction endonuclease HinfI, using a simple PCR procedure to synthesize DNA of known sequence in which every cytosine is methylated at the 5 position. We find that HinfI cannot digest cytosine methylated DNA at the concentrations normally used in restriction digests. Complete digestion is possible using a vast excess of enzyme; under these conditions, the rate of HinfI digestion for cytosine methylated DNA is at least 1440-fold slower than for unmethylated DNA. The presence of an additional methylated cytosine at the degenerate position internal to the recognition sequence does not appear to increase the resistance to HinfI digestion. We also tested HhaII, an isoschizomer of HinfI, and found that it is completely inactive on cytosine methylated DNA. The procedure we have used should be of general applicability in determination of the methylation sensitivities of other restiction enzymes, as well as studies of the effects of methylation on gene expression in direct DNA transfer experiments.

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The A and A* proteins of phage phi X174 are encoded in the same reading frame in the viral genome; the smaller A protein is the result of a translational start signal with the A gene. To differentiate their respective functions, oligonucleotide-directed site-specific mutagenesis was used to change the ATG start codon of the phi X 174 A* gene, previously cloned into pCQV2 under lambda repressor control, into a TAG stop codon. The altered A gene was then inserted back into phi X replicative form DNA to produce an amber mutant, phi XamA*. Two different Escherichia coli amber suppressor strains infected with this mutant produced viable progeny phage with only a slight reduction in yield. In Su+ cells infected with phi XamA*, phi X gene A protein, altered at one amino acid, was synthesized at normal levels; A* protein was not detectable. These observations indicate that the A* protein increases the replicative efficiency of the phage, perhaps by shutting down host DNA replication, but is not required for replication of phi X174 DNA or the packaging of the viral strand under the conditions tested.

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Recombinant DNA techniques were used to study various aspects of rep gene function in Escherichia coli. In order to enhance expression of the Rep protein, the rep gene was cloned into the vector pKC30 under the control of the lambda pL promoter. By trimming away a portion of the DNA sequence immediately upstream of the translational start site of rep, we were able to obtain very high levels of Rep protein upon induction. Cells carrying such plasmids showed no ill effects from the high concentration of the protein. To ascertain the consequence of the absence of Rep protein on the cell, the chromosomal copy of the gene was deleted using a homologous recombination technique. The viability of E. coli strains completely lacking the rep gene proves that the Rep function is not essential, at least in wild-type cells under laboratory conditions. We confirmed that in the absence of Rep function there is an increase in the average number of growing forks in exponentially growing cells; augmentation of Rep protein levels above normal, however, did not detectably decrease the number of growing forks.

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The A* gene of bacteriophage phi X174 has been cloned into the inducible expression vector pCQV2 under conditions allowing its lethal action to be controlled by the lambda cI857 repressor. Upon induction of expression, DNA synthesis in Escherichia coli carrying the recombinant plasmid is severely inhibited; however, these same cells permit beta-galactosidase induction at a rate similar to that observed in control cells at the inducing (for A*) temperature. Cells in which A* is expressed form filaments and produce more RecA protein, indicating at least a partial induction of the SOS response; however, there is no evidence of damage to the bacterial chromosome. It appears that the A* protein has as one function the inhibition of cell division and DNA replication but not transcription or protein synthesis during phage infection.

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