Timothy Kelliher

Hypoxia Triggers Meiotic Fate Acquisition in Maize

Redox Status Incites Gametogenesis Germ cells differ from somatic cells in their chromosomal complement, being haploid rather than diploid. In animals, the germ cells are generally produced by a separate lineage set aside early in development. Plants, however, lack a reserved germ cell lineage. Kelliher and Walbot (p. 345; see the Perspective by Whipple) now show that, in maize, the key signal for germ cell production is hypoxia, which triggers differentiation of anther germ cells from a generalized field of progenitors. The specializing germ cells then induce differentiation of supportive somatic cells. Maize anthers use cellular redox status rather than a specific germ cell lineage to signal production of new germ cells. Evidence from confocal microscopic reconstruction of maize anther development in fertile, mac1 (excess germ cells), and msca1 (no germ cells) flowers indicates that the male germ line is multiclonal and uses the MAC1 protein to organize the somatic niche. Furthermore, we identified redox status as a determinant of germ cell fate, defining a mechanism distinct from the animal germ cell lineage. Decreasing oxygen or H2O2 increases germ cell numbers, stimulates superficial germ cell formation, and rescues germinal differentiation in msca1 flowers. Conversely, oxidizing environments inhibit germ cell specification and cause ectopic differentiation in deeper tissues. We propose that hypoxia, arising naturally within growing anther tissue, acts as a positional cue to set germ cell fate.

Maize multiple archesporial cells 1 (mac1), an ortholog of rice TDL1A, modulates cell proliferation and identity in early anther development

To ensure fertility, complex somatic and germinal cell proliferation and differentiation programs must be executed in flowers. Loss-of-function of the maize multiple archesporial cells 1 (mac1) gene increases the meiotically competent population and ablates specification of somatic wall layers in anthers. We report the cloning of mac1, which is the ortholog of rice TDL1A. Contrary to prior studies in rice and Arabidopsis in which mac1-like genes were inferred to act late to suppress trans-differentiation of somatic tapetal cells into meiocytes, we find that mac1 anthers contain excess archesporial (AR) cells that proliferate at least twofold more rapidly than normal prior to tapetal specification, suggesting that MAC1 regulates cell proliferation. mac1 transcript is abundant in immature anthers and roots. By immunolocalization, MAC1 protein accumulates preferentially in AR cells with a declining radial gradient that could result from diffusion. By transient expression in onion epidermis, we demonstrate experimentally that MAC1 is secreted, confirming that the predicted signal peptide domain in MAC1 leads to secretion. Insights from cytology and double-mutant studies with ameiotic1 and absence of first division1 mutants confirm that MAC1 does not affect meiotic cell fate; it also operates independently of an epidermal, Ocl4-dependent pathway that regulates proliferation of subepidermal cells. MAC1 both suppresses excess AR proliferation and is responsible for triggering periclinal division of subepidermal cells. We discuss how MAC1 can coordinate the temporal and spatial pattern of cell proliferation in maize anthers.

Maize transformation technology development for commercial event generation

Maize is an important food and feed crop in many countries. It is also one of the most important target crops for the application of biotechnology. Currently, there are more biotech traits available on the market in maize than in any other crop. Generation of transgenic events is a crucial step in the development of biotech traits. For commercial applications, a high throughput transformation system producing a large number of high quality events in an elite genetic background is highly desirable. There has been tremendous progress in Agrobacterium-mediated maize transformation since the publication of the Ishida et al. (1996) paper and the technology has been widely adopted for transgenic event production by many labs around the world. We will review general efforts in establishing efficient maize transformation technologies useful for transgenic event production in trait research and development. The review will also discuss transformation systems used for generating commercial maize trait events currently on the market. As the number of traits is increasing steadily and two or more modes of action are used to control key pests, new tools are needed to efficiently transform vectors containing multiple trait genes. We will review general guidelines for assembling binary vectors for commercial transformation. Approaches to increase transformation efficiency and gene expression of large gene stack vectors will be discussed. Finally, recent studies of targeted genome modification and transgene insertion using different site-directed nuclease technologies will be reviewed.

MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction.

Sexual reproduction in flowering plants involves double fertilization, the union of two sperm from pollen with two sex cells in the female embryo sac. Modern plant breeders increasingly seek to circumvent this process to produce doubled haploid individuals, which derive from the chromosome-doubled cells of the haploid gametophyte. Doubled haploid production fixes recombinant haploid genomes in inbred lines, shaving years off the breeding process. Costly, genotype-dependent tissue culture methods are used in many crops, while seed-based in vivo doubled haploid systems are rare in nature and difficult to manage in breeding programmes. The multi-billion-dollar maize hybrid seed business, however, is supported by industrial doubled haploid pipelines using intraspecific crosses to in vivo haploid inducer males derived from Stock 6, first reported in 1959 (ref. 5), followed by colchicine treatment. Despite decades of use, the mode of action remains controversial. Here we establish, through fine mapping, genome sequencing, genetic complementation, and gene editing, that haploid induction in maize (Zea mays) is triggered by a frame-shift mutation in MATRILINEAL (MTL), a pollen-specific phospholipase, and that novel edits in MTL lead to a 6.7% haploid induction rate (the percentage of haploid progeny versus total progeny). Wild-type MTL protein localizes exclusively to sperm cytoplasm, and pollen RNA-sequence profiling identifies a suite of pollen-specific genes overexpressed during haploid induction, some of which may mediate the formation of haploid seed. These findings highlight the importance of male gamete cytoplasmic components to reproductive success and male genome transmittance. Given the conservation of MTL in the cereals, this discovery may enable development of in vivo haploid induction systems to accelerate breeding in crop plants.

Maize germinal cell initials accommodate hypoxia and precociously express meiotic genes.

In flowering plants, anthers are the site of de novo germinal cell specification, male meiosis, and pollen development. Atypically, anthers lack a meristem. Instead, both germinal and somatic cell types differentiate from floral stem cells packed into anther lobes. To better understand anther cell fate specification and to provide a resource for the reproductive biology community, we isolated cohorts of germinal and somatic initials from maize anthers within 36 h of fate acquisition, identifying 815 specific and 1714 significantly enriched germinal transcripts, plus 2439 specific and 2112 significantly enriched somatic transcripts. To clarify transcripts involved in cell differentiation, we contrasted these profiles to anther primordia prior to fate specification and to msca1 anthers arrested in the first step of fate specification and hence lacking normal cell types. The refined cell-specific profiles demonstrated that both germinal and somatic cell populations differentiate quickly and express unique transcription factor sets; a subset of transcript localizations was validated by in situ hybridization. Surprisingly, germinal initials starting 5 days of mitotic divisions were enriched significantly in >100 transcripts classified in meiotic processes that included recombination and synapsis, along with gene sets involved in RNA metabolism, redox homeostasis, and cytoplasmic ATP generation. Enrichment of meiotic-specific genes in germinal initials challenges current dogma that the mitotic to meiotic transition occurs later in development during pre-meiotic S phase. Expression of cytoplasmic energy generation genes suggests that male germinal cells accommodate hypoxia by diverting carbon away from mitochondrial respiration into alternative pathways that avoid producing reactive oxygen species (ROS).

Maternal Haploids Are Preferentially Induced by CENH3-tailswap Transgenic Complementation in Maize

Doubled haploid plants are invaluable breeding tools but many crop species are recalcitrant to available haploid induction techniques. To test if haploid inducer lines can be engineered into crops, CENH3−∕− and CENH3:RNAi lines were complemented by AcGREEN-tailswap-CENH3 or AcGREEN-CENH3 transgenes. Haploid induction rates were determined following testcrosses to wild-type plants after independently controlling for inducer parent sex and transgene zygosity. CENH3 fusion proteins were localized to centromeres and did not cause vegetative defects or male sterility. CENH3:RNAi lines did not demonstrate consistent knockdown and rarely produced haploids. In contrast, many of the complemented CENH3−∕− lines produced haploids at low frequencies. The rate of gynogenic haploid induction reached a maximum of 3.6% in several hemizygous individuals when backcrossed as males. These results demonstrate that CENH3-tailswap transgenes can be used to engineer in vivo haploid induction systems into maize plants.

Transcriptomes and Proteomes Define Gene Expression Progression in Pre-meiotic Maize Anthers

Plants lack a germ line; consequently, during reproduction adult somatic cells within flowers must switch from mitotic proliferation to meiosis. In maize (Zea mays L.) anthers, hypoxic conditions in the developing tassel trigger pre-meiotic competence in the column of pluripotent progenitor cells in the center of anther lobes, and within 24 hr these newly specified germinal cells have patterned their surrounding neighbors to differentiate as the first somatic niche cells. Transcriptomes were analyzed by microarray hybridization in carefully staged whole anthers during initial specification events, after the separation of germinal and somatic lineages, during the subsequent rapid mitotic proliferation phase, and during final pre-meiotic germinal and somatic cell differentiation. Maize anthers exhibit a highly complex transcriptome constituting nearly three-quarters of annotated maize genes, and expression patterns are dynamic. Laser microdissection was applied to begin assigning transcripts to tissue and cell types and for comparison to transcriptomes of mutants defective in cell fate specification. Whole anther proteomes were analyzed at three developmental stages by mass spectrometric peptide sequencing using size-fractionated proteins to evaluate the timing of protein accumulation relative to transcript abundance. New insights include early and sustained expression of meiosis-associated genes (77.5% of well-annotated meiosis genes are constitutively active in 0.15 mm anthers), an extremely large change in transcript abundances and types a few days before meiosis (including a class of 1340 transcripts absent specifically at 0.4 mm), and the relative disparity between transcript abundance and protein abundance at any one developmental stage (based on 1303 protein-to-transcript comparisons).

Unresolved issues in pre-meiotic anther development

Compared to the diversity of other floral organs, the steps in anther ontogeny, final cell types, and overall organ shape are remarkably conserved among Angiosperms. Defects in pre-meiotic anthers that alter cellular composition or function typically result in male-sterility. Given the ease of identifying male-sterile mutants, dozens of genes with key roles in early anther development have been identified and cloned in model species, ordered by time of action and spatiotemporal expression, and used to propose explanatory models for critical steps in cell fate specification. Despite rapid progress, fundamental issues in anther development remain unresolved, and it is unclear if insights from one species can be applied to others. Here we construct a comparison of Arabidopsis, rice, and maize immature anthers to pinpoint distinctions in developmental pace. We analyze the mechanisms by which archesporial (pre-meiotic) cells are specified distinct from the soma, discuss what constitutes meiotic preparation, and review what is known about the secondary parietal layer and its terminal periclinal division that generates the tapetal and middle layers. Finally, roles for small RNAs are examined, focusing on the grass-specific phasiRNAs.

OsMATL mutation induces haploid seed formation in indica rice

Intraspecific haploid induction in maize (Zea mays) is triggered by a native frameshift mutation in MATRILINEAL (MATL), which encodes a pollen-specific phospholipase. To develop a haploid inducer in rice (Oryza sativa), we generated an allelic series in the putative ZmMATL orthologue, OsMATL, and found that knockout mutations led to a reduced seed set and a 2–6% haploid induction rate. This demonstrates MATL functional conservation and represents a major advance for rice breeding.A MATL gene mutation was found to induce haploids in maize. Now, knocking out the MATL orthologue in rice results in haploid induction at a rate of 2–6%, suggesting the functional conservation of MATL, and represents an advance for rice breeding.