Concerted modification of flowering time and inflorescence architecture by ectopic expression of TFL1-like genes in maize.

TERMINAL FLOWER1 (TFL1)-like genes are highly conserved in plants and are thought to function in the maintenance of meristem indeterminacy. Recently, we described six maize (Zea mays) TFL1-related genes, named ZEA CENTRORADIALIS1 (ZCN1) to ZCN6. To gain insight into their functions, we generated transgenic maize plants overexpressing their respective cDNAs driven by a constitutive promoter. Overall, ectopic expression of the maize TFL1-like genes produced similar phenotypes, including delayed flowering and altered inflorescence architecture. We observed an apparent relationship between the magnitude of the transgenic phenotypes and the degree of homology between the ZCN proteins. ZCN2, -4, and -5 form a monophylogenetic clade, and their overexpression produced the strongest phenotypes. Along with very late flowering, these transgenic plants produced a "bushy" tassel with increased lateral branching and spikelet density compared with nontransgenic siblings. On the other hand, ZCN1, -3, and -6 produced milder effects. Among them, ZCN1 showed moderate effects on flowering time and tassel morphology, whereas ZCN3 and ZCN6 did not change flowering time but still showed effects on tassel morphology. In situ hybridizations of tissue from nontransgenic plants revealed that the expression of all ZCN genes was associated with vascular bundles, but each gene had a specific spatial and temporal pattern. Expression of four ZCN genes localized to the protoxylem, whereas ZCN5 was expressed in the protophloem. Collectively, our findings suggest that ectopic expression of the TFL1-like genes in maize modifies flowering time and inflorescence architecture through maintenance of the indeterminacy of the vegetative and inflorescence meristems.

Artificial chromosome formation in maize (Zea mays L.).

We report on the construction of maize minichromosomes using shuttle vectors harboring native centromeric segments, origins of replication, selectable marker genes, and telomeric repeats. These vectors were introduced into scutellar cells of maize immature embryos by microprojectile bombardment. Several independent transformation events were identified containing minichromosomes in addition to the normal diploid complement of 20 maize chromosomes. Immunostaining indicated that the minichromosomes recruited centromeric protein C, which is a specific component of the centromere/kinetochore complex. Minichromosomes were estimated to be 15-30 Mb in size based on cytological measurements. Fluorescent in situ hybridization (FISH) showed that minichromosomes contain the centromeric, telomeric, and exogenous unique marker sequences interspersed with maize retrotransposons. Minichromosomes were detected for at least a year in actively dividing callus cultures, providing evidence for their stability through numerous cell cycles. Plants were regenerated and minichromosomes were detected in root tips, providing confirmation of their normal replication and transmission during mitosis and through organogenesis. Assembly of maize artificial chromosomes may provide a tool to study centromere function and a foundation for developing new high capacity vectors for plant functional genomics and breeding.

Involvement of the MADS-box gene ZMM4 in floral induction and inflorescence development in maize.

The switch from vegetative to reproductive growth is marked by the termination of vegetative development and the adoption of floral identity by the shoot apical meristem (SAM). This process is called the floral transition. To elucidate the molecular determinants involved in this process, we performed genome-wide RNA expression profiling on maize (Zea mays) shoot apices at vegetative and early reproductive stages using massively parallel signature sequencing technology. Profiling revealed significant up-regulation of two maize MADS-box (ZMM) genes, ZMM4 and ZMM15, after the floral transition. ZMM4 and ZMM15 map to duplicated regions on chromosomes 1 and 5 and are linked to neighboring MADS-box genes ZMM24 and ZMM31, respectively. This gene order is syntenic with the vernalization1 locus responsible for floral induction in winter wheat (Triticum monococcum) and similar loci in other cereals. Analyses of temporal and spatial expression patterns indicated that the duplicated pairs ZMM4-ZMM24 and ZMM15-ZMM31 are coordinately activated after the floral transition in early developing inflorescences. More detailed analyses revealed ZMM4 expression initiates in leaf primordia of vegetative shoot apices and later increases within elongating meristems acquiring inflorescence identity. Expression analysis in late flowering mutants positioned all four genes downstream of the floral activators indeterminate1 (id1) and delayed flowering1 (dlf1). Overexpression of ZMM4 leads to early flowering in transgenic maize and suppresses the late flowering phenotype of both the id1 and dlf1 mutations. Our results suggest ZMM4 may play roles in both floral induction and inflorescence development.

A genomic and expression compendium of the expanded PEBP gene family from maize.

The phosphatidylethanolamine-binding proteins (PEBPs) represent an ancient protein family found across the biosphere. In animals they are known to act as kinase and serine protease inhibitors controlling cell growth and differentiation. In plants the most extensively studied PEBP genes, the Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) genes, function, respectively, as a promoter and a repressor of the floral transition. Twenty-five maize (Zea mays) genes that encode PEBP-like proteins, likely the entire gene family, were identified and named Zea mays CENTRORADIALIS (ZCN), after the first described plant PEBP gene from Antirrhinum. The maize family is expanded relative to eudicots (typically six to eight genes) and rice (Oryza sativa; 19 genes). Genomic structures, map locations, and syntenous relationships with rice were determined for 24 of the maize ZCN genes. Phylogenetic analysis assigned the maize ZCN proteins to three major subfamilies: TFL1-like (six members), MOTHER OF FT AND TFL1-like (three), and FT-like (15). Expression analysis demonstrated transcription for at least 21 ZCN genes, many with developmentally specific patterns and some having alternatively spliced transcripts. Expression patterns and protein structural analysis identified maize candidates likely having conserved gene function of TFL1. Expression patterns and interaction of the ZCN8 protein with the floral activator DLF1 in the yeast (Saccharomyces cerevisiae) two-hybrid assay strongly supports that ZCN8 plays an orthologous FT function in maize. The expression of other ZCN genes in roots, kernels, and flowers implies their involvement in diverse developmental processes.

Conserved noncoding genomic sequences associated with a flowering-time quantitative trait locus in maize.

Flowering time is a fundamental trait of maize adaptation to different agricultural environments. Although a large body of information is available on the map position of quantitative trait loci for flowering time, little is known about the molecular basis of quantitative trait loci. Through positional cloning and association mapping, we resolved the major flowering-time quantitative trait locus, Vegetative to generative transition 1 (Vgt1), to an approximately 2-kb noncoding region positioned 70 kb upstream of an Ap2-like transcription factor that we have shown to be involved in flowering-time control. Vgt1 functions as a cis-acting regulatory element as indicated by the correlation of the Vgt1 alleles with the transcript expression levels of the downstream gene. Additionally, within Vgt1, we identified evolutionarily conserved noncoding sequences across the maize-sorghum-rice lineages. Our results support the notion that changes in distant cis-acting regulatory regions are a key component of plant genetic adaptation throughout breeding and evolution.

delayed flowering1 Encodes a basic leucine zipper protein that mediates floral inductive signals at the shoot apex in maize.

Separation of the life cycle of flowering plants into two distinct growth phases, vegetative and reproductive, is marked by the floral transition. The initial floral inductive signals are perceived in the leaves and transmitted to the shoot apex, where the vegetative shoot apical meristem is restructured into a reproductive meristem. In this study, we report cloning and characterization of the maize (Zea mays) flowering time gene delayed flowering1 (dlf1). Loss of dlf1 function results in late flowering, indicating dlf1 is required for timely promotion of the floral transition. dlf1 encodes a protein with a basic leucine zipper domain belonging to an evolutionarily conserved family. Three-dimensional protein modeling of a missense mutation within the basic domain suggests DLF1 protein functions through DNA binding. The spatial and temporal expression pattern of dlf1 indicates a threshold level of dlf1 is required in the shoot apex for proper timing of the floral transition. Double mutant analysis of dlf1 and indeterminate1 (id1), another late flowering mutation, places dlf1 downstream of id1 function and suggests dlf1 mediates floral inductive signals transmitted from leaves to the shoot apex. This study establishes an emergent framework for the genetic control of floral induction in maize and highlights the conserved topology of the floral transition network in flowering plants.

Duplicated fie genes in maize: expression pattern and imprinting suggest distinct functions.

Two maize genes with predicted translational similarity to the Arabidopsis FIE (Fertilization-Independent Endosperm) protein, a repressor of endosperm development in the absence of fertilization, were cloned and analyzed. Genomic sequences of fie1 and fie2 show significant homology within coding regions but none within introns or 5' upstream. The fie1 gene is expressed exclusively in the endosperm of developing kernels starting at approximately 6 days after pollination. fie1 is an imprinted gene showing no detectable expression of the paternally derived fie1 allele during kernel development. Conversely, fie2 is expressed in the embryo sac before pollination. After pollination, its expression persists, predominantly in the embryo and at lower levels in the endosperm. The paternal fie2 allele is not expressed early in kernel development, but its transcription is activated at 5 days after pollination. fie2 is likely to be a functional ortholog of the Arabidopsis FIE gene, whereas fie1 has evolved a distinct function. The maize FIE2 and sorghum FIE proteins form a monophyletic group, sharing a closer relationship to each other than to the FIE1 protein, suggesting that maize fie genes originated from two different ancestral genomes.

Isolation of A/D and C genome specific dispersed and clustered repetitive DNA sequences from Avena sativa.

DNA gel-blot and in situ hybridization with genome-specific repeated sequences have proven to be valuable tools in analyzing genome structure and relationships in species with complex allopolyploid genomes such as hexaploid oat (Avena sativa L., 2n = 6x = 42; AACCDD genome). In this report, we describe a systematic approach for isolating genome-, chromosome-, and region-specific repeated and low-copy DNA sequences from oat that can presumably be applied to any complex genome species. Genome-specific DNA sequences were first identified in a random set of A. sativa genomic DNA cosmid clones by gel-blot hybridization using labeled genomic DNA from different Avena species. Because no repetitive sequences were identified that could distinguish between the A and D gneomes, sequences specific to these two genomes are refereed to as A/D genome specific. A/D or C genome specific DNA subfragments were used as screening probes to identify additional genome-specific cosmid clones in the A. sativa genomic library. We identified clustered and dispersed repetitive DNA elements for the A/D and C genomes that could be used as cytogenetic markers for discrimination of the various oat chromosomes. Some analyzed cosmids appeared to be composed entirely of genome-specific elements, whereas others represented regions with genome- and non-specific repeated sequences with interspersed low-copy DNA sequences. Thus, genome-specific hybridization analysis of restriction digests of random and selected A. sativa cosmids also provides insight into the sequence organization of the oat genome.