In the Gramineae, the cyclic hydroxamic acids 2,4-dihydroxy-1, 4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1, 4-benzoxazin-3-one (DIMBOA) form part of the defense against insects and microbial pathogens. Five genes, Bx1 through Bx5, are required for DIBOA biosynthesis in maize. The functions of these five genes, clustered on chromosome 4, were demonstrated in vitro. Bx1 encodes a tryptophan synthase alpha homolog that catalyzes the formation of indole for the production of secondary metabolites rather than tryptophan, thereby defining the branch point from primary to secondary metabolism. Bx2 through Bx5 encode cytochrome P450-dependent monooxygenases that catalyze four consecutive hydroxylations and one ring expansion to form the highly oxidized DIBOA.

Central America (1), natural genetic variations in flowering time enabled early Native Americans to select maize adapted to a range of latitudes and lengths of growing seasons, including the very short summer season typical of the eastern Canadian region of Quebec. Under such conditions, early flowering allows seed to mature before the onset of frost. Flowering time is also a key trait of improved drought tolerance. Indeed, it has been shown that a single day of drought during flowering can decrease yield by as much as 8% (2). One way to address such losses is to develop and grow cultivars characterized by a short cycle and able to flower before predictable drought episodes.The genetic variability available for maize breeding is essentially quantitative; i.e., it involves allelic variation at different quantitative trait loci (QTLs), which are influenced by environmental effects. Although a large body of mapping information on QTLs is available for flowering time (3), relatively little is known about the molecular basis of QTLs, with only one gene, Dwarf8, correlated thus far with quantitative effects (4, 5). Furthermore, a few mutants for flowering time have been described (6, 7), two of which, id1 (8) and dlf1 (9), have been cloned. Our results (i) show that the allelic variation responsible for the major flowering-time QTL, Vegetative to generative transition 1 (Vgt1) (10, 11) on chromosome 8, is confined to an Ϸ2-kb intergenic region upstream of an Ap2-like flowering-time gene, (ii) identify maize-sorghum-rice evolutionarily conserved noncoding sequences (CNSs) within Vgt1, and (iii) support a cisacting transcription-regulatory role for Vgt1. ResultsPositional Cloning of Vgt1. Previous work (12) mapped Vgt1 to a 1.3-cM region (Fig. 1A) on bin 8.05, based on a mapping population derived from the cross N28 ϫ C22-4. The strain C22-4 is nearly isogenic to N28 and carries the early Vgt1 allele in an Ϸ7-cM introgression originating from the early maize variety Gaspé Flint. By using standard positional cloning, Vgt1 was confined to an Ϸ2-kb region (Fig. 1 B-D). Sequence annotation of the original BAC clone and the corresponding sequences derived from N28 and Gaspé Flint genetic backgrounds showed that Vgt1 is apparently noncoding and is located Ϸ70 kb (61-76 kb, depending on the genetic background) upstream of an Ap2-like gene identified here as ZmRap2.7. This gene is orthologous to Rap2.7 (also known as TOE1), a transcription factor that regulates flowering time in Arabidopsis (13,14). No other genes were annotated between Vgt1 and ZmRap2.7. Pseudogenes due to transduplication events mediated by nonautonomous helitron elements (15) were observed in N28 and other genetic backgrounds but not in Gaspé Flint (data not shown). Within the Vgt1 region, the contrasting QTL alleles showed 29 SNPs and insertion/deletion-type polymorphisms (Indels) and one 143-bp insertion into the Gaspé Flint allele of a Mite transposon belonging to the Tourist (16) family [ Fig. 4 Lower and supporting information (SI) Fig. 5].Association M...

In maize (Zea mays), sex determination occurs through abortion of female carpels in the tassel and arrest of male stamens in the ear. The Tasselseed6 (Ts6) and tasselseed4 (ts4) mutations permit carpel development in the tassel while increasing meristem branching, showing that sex determination and acquisition of meristem fate share a common pathway. We show that ts4 encodes a mir172 microRNA that targets APETALA2 floral homeotic transcription factors. Three lines of evidence suggest that indeterminate spikelet1 (ids1), an APETALA2 gene required for spikelet meristem determinacy, is a key target of ts4. First, loss of ids1 suppresses the ts4 sex determination and branching defects. Second, Ts6 mutants phenocopy ts4 and possess mutations in the microRNA binding site of ids1. Finally, IDS1 protein is expressed more broadly in ts4 mutants compared to wild type. Our results demonstrate that sexual identity in maize is acquired by limiting floral growth through negative regulation of the floral homeotic pathway.

The architecture of higher plants is established through the activity of lateral meristems--small groups of stem cells formed during vegetative and reproductive development. Lateral meristems generate branches and inflorescence structures, which define the overall form of a plant, and are largely responsible for the evolution of different plant architectures. Here, we report the isolation of the barren stalk1 gene, which encodes a non-canonical basic helix-loop-helix protein required for the initiation of all aerial lateral meristems in maize. barren stalk1 represents one of the earliest genes involved in the patterning of maize inflorescences, and, together with the teosinte branched1 gene, it regulates vegetative lateral meristem development. The architecture of maize has been a major target of selection for early agriculturalists and modern farmers, because it influences harvesting, breeding strategies and mechanization. By sampling nucleotide diversity in the barren stalk1 region, we show that two haplotypes entered the maize gene pool from its wild progenitor, teosinte, and that only one was incorporated throughout modern inbreds, suggesting that barren stalk1 was selected for agronomic purposes.

Phytic acid in cereal grains and oilseeds is poorly digested by monogastric animals and negatively affects animal nutrition and the environment. However, breeding programs involving mutants with less phytic acid and more inorganic phosphate (P(i)) have been frustrated by undesirable agronomic characteristics associated with the phytic acid-reducing mutations. We show that maize lpa1 mutants are defective in a multidrug resistance-associated protein (MRP) ATP-binding cassette (ABC) transporter that is expressed most highly in embryos, but also in immature endosperm, germinating seed and vegetative tissues. Silencing expression of this transporter in an embryo-specific manner produced low-phytic-acid, high-Pi transgenic maize seeds that germinate normally and do not show any significant reduction in seed dry weight. This dominant transgenic approach obviates the need for incorporating recessive lpa1 mutations to create maize hybrids with reduced phytic acid. Suppressing the homologous soybean MRP gene also generated low-phytic-acid seed, suggesting that the strategy might be feasible for many crops.

Endosperm of cereal grains is one of the most important renewable resources for food, feed, and industrial raw material. It consists of four triploid cell types, i.e., aleurone, starchy endosperm, transfer cells, and cells of the embryo surrounding region. In maize, the aleurone layer is one cell layer thick and covers most of the perimeter of the endosperm. Specification of maize aleurone cell fate is proposed to occur through activation of the tumor necrosis factor receptor-like receptor kinase CRINKLY4. A second maize gene essential for aleurone cell development is defective kernel 1 (dek1). Here we show that DEK1 shares high homology with animal calpains. The predicted 2,159-aa DEK1 protein has 21 transmembrane regions, an extracellular loop, and a cysteine proteinase domain that shares high homology with domain II of m-calpain from animals. We propose that DEK1 functions to maintain and restrict the aleurone cell fate imposed by CR4 through activation of its cysteine proteinase by contact with the outer endosperm surface. DEK1 seems to be the only member of the calpain superfamily in plants, Arabidopsis DEK1 sharing 70% overall identity with maize DEK1. The expression of dek1 in most plant tissues in maize and Arabidopsis, as well as its presence in a variety of higher plants, including angiosperms and gymnosperms, suggests that DEK1 plays a conserved role in plant signal transduction.

Apomixis is a form of asexual reproduction through seeds in angiosperms. Apomictic plants bypass meiosis and fertilization, developing offspring that are genetically identical to their mother. In a genetic screen for maize (Zea mays) mutants mimicking aspects of apomixis, we identified a dominant mutation resulting in the formation of functional unreduced gametes. The mutant shows defects in chromatin condensation during meiosis and subsequent failure to segregate chromosomes. The mutated locus codes for AGO104, a member of the ARGONAUTE family of proteins. AGO104 accumulates specifically in somatic cells surrounding the female meiocyte, suggesting a mobile signal rather than cellautonomous control. AGO104 is necessary for non-CG methylation of centromeric and knob-repeat DNA. Digital gene expression tag profiling experiments using high-throughput sequencing show that AGO104 influences the transcription of many targets in the ovaries, with a strong effect on centromeric repeats. AGO104 is related to Arabidopsis thaliana AGO9, but while AGO9 acts to repress germ cell fate in somatic tissues, AGO104 acts to repress somatic fate in germ cells. Our findings show that female germ cell development in maize is dependent upon conserved small RNA pathways acting noncell-autonomously in the ovule. Interfering with this repression leads to apomixis-like phenotypes in maize.

The orderly production of meristems with specific fates is crucial for the proper elaboration of plant architecture. The maize inflorescence meristem branches several times to produce lateral meristems with determinate fates. The first meristem formed, the spikelet pair meristem, produces two spikelet meristems, each of which produces two floral meristems. We have identified a gene called indeterminate spikelet1 (ids1) that specifies a determinate spikelet meristem fate and thereby limits the number of floral meristems produced. In the absence of ids1 gene function, the spikelet meristem becomes indeterminate and produces additional florets. Members of the grass family vary in the number of florets within their spikelets, suggesting that ids1 may play a role in inflorescence architecture in other grass species. ids1 is a member of the APETALA2 (AP2) gene family of transcription factors that has been implicated in a wide range of plant development roles. Expression of ids1 was detected in many types of lateral organ primordia as well as spikelet meristems. Our analysis of the ids1 mutant phenotype and expression pattern indicates that ids1 specifies determinate fates by suppressing indeterminate growth within the spikelet meristem.