Darren J. Morrow

Sequencing, mapping, and analysis of 27,455 maize full-length cDNAs.

Full-length cDNA (FLcDNA) sequencing establishes the precise primary structure of individual gene transcripts. From two libraries representing 27 B73 tissues and abiotic stress treatments, 27,455 high-quality FLcDNAs were sequenced. The average transcript length was 1.44 kb including 218 bases and 321 bases of 5′ and 3′ UTR, respectively, with 8.6% of the FLcDNAs encoding predicted proteins of fewer than 100 amino acids. Approximately 94% of the FLcDNAs were stringently mapped to the maize genome. Although nearly two-thirds of this genome is composed of transposable elements (TEs), only 5.6% of the FLcDNAs contained TE sequences in coding or UTR regions. Approximately 7.2% of the FLcDNAs are putative transcription factors, suggesting that rare transcripts are well-enriched in our FLcDNA set. Protein similarity searching identified 1,737 maize transcripts not present in rice, sorghum, Arabidopsis, or poplar annotated genes. A strict FLcDNA assembly generated 24,467 non-redundant sequences, of which 88% have non-maize protein matches. The FLcDNAs were also assembled with 41,759 FLcDNAs in GenBank from other projects, where semi-strict parameters were used to identify 13,368 potentially unique non-redundant sequences from this project. The libraries, ESTs, and FLcDNA sequences produced from this project are publicly available. The annotated EST and FLcDNA assemblies are available through the maize FLcDNA web resource (www.maizecdna.org).

Comparative profiling of the sense and antisense transcriptome of maize lines

BackgroundThere are thousands of maize lines with distinctive normal as well as mutant phenotypes. To determine the validity of comparisons among mutants in different lines, we first address the question of how similar the transcriptomes are in three standard lines at four developmental stages.ResultsFour tissues (leaves, 1 mm anthers, 1.5 mm anthers, pollen) from one hybrid and one inbred maize line were hybridized with the W23 inbred on Agilent oligonucleotide microarrays with 21,000 elements. Tissue-specific gene expression patterns were documented, with leaves having the most tissue-specific transcripts. Haploid pollen expresses about half as many genes as the other samples. High overlap of gene expression was found between leaves and anthers. Anther and pollen transcript expression showed high conservation among the three lines while leaves had more divergence. Antisense transcripts represented about 6 to 14 percent of total transcriptome by tissue type but were similar across lines. Gene Ontology (GO) annotations were assigned and tabulated. Enrichment in GO terms related to cell-cycle functions was found for the identified antisense transcripts. Microarray results were validated via quantitative real-time PCR and by hybridization to a second oligonucleotide microarray platform.ConclusionDespite high polymorphisms and structural differences among maize inbred lines, the transcriptomes of the three lines displayed remarkable similarities, especially in both reproductive samples (anther and pollen). We also identified potential stage markers for maize anther development. A large number of antisense transcripts were detected and implicated in important biological functions given the enrichment of particular GO classes.

Genome-wide mutagenesis of Zea mays L. using RescueMu transposons

Derived from the maize Mu1 transposon, RescueMu provides strategies for maize gene discovery and mutant phenotypic analysis. 9.92 Mb of gene-enriched sequences next to RescueMu insertion sites were co-assembled with expressed sequence tags and analyzed. Multiple plasmid recoveries identified probable germinal insertions and screening of RescueMu plasmid libraries identified plants containing probable germinal insertions. Although frequently recovered parental insertions and insertion hotspots reduce the efficiency of gene discovery per plasmid, RescueMu targets a large variety of genes and produces knockout mutants.

Transcriptomic, proteomic and metabolomic analysis of UV-B signaling in maize

BackgroundUnder normal solar fluence, UV-B damages macromolecules, but it also elicits physiological acclimation and developmental changes in plants. Excess UV-B decreases crop yield. Using a treatment twice solar fluence, we focus on discovering signals produced in UV-B-irradiated maize leaves that translate to systemic changes in shielded leaves and immature ears.ResultsUsing transcriptome and proteomic profiling, we tracked the kinetics of transcript and protein alterations in exposed and shielded organs over 6 h. In parallel, metabolic profiling identified candidate signaling molecules based on rapid increase in irradiated leaves and increased levels in shielded organs; pathways associated with the synthesis, sequestration, or degradation of some of these potential signal molecules were UV-B-responsive. Exposure of just the top leaf substantially alters the transcriptomes of both irradiated and shielded organs, with greater changes as additional leaves are irradiated. Some phenylpropanoid pathway genes are expressed only in irradiated leaves, reflected in accumulation of pathway sunscreen molecules. Most protein changes detected occur quickly: approximately 92% of the proteins in leaves and 73% in immature ears changed after 4 h UV-B were altered by a 1 h UV-B treatment.ConclusionsThere were significant transcriptome, proteomic, and metabolomic changes under all conditions studied in both shielded and irradiated organs. A dramatic decrease in transcript diversity in irradiated and shielded leaves occurs between 0 h and 1 h, demonstrating the susceptibility of plants to short term UV-B spikes as during ozone depletion. Immature maize ears are highly responsive to canopy leaf exposure to UV-B.

Distinctive transcriptome responses to adverse environmental conditions in Zea mays L.

SUMMARY Maize seedling transcriptome responses to six abiotic perturbations (heat, cold, darkness, desiccation, salt, ultraviolet-B) were analysed. Approximately 7800 transcripts were expressed in one or more treatments compared with light-grown seedlings plus juvenile leaves from field-grown plants. Approximately 5200 transcripts were expressed in one or more treatments and absent in light-grown seedlings. Approximately 2000 transcripts were unique to one treatment. Salt and heat elicited the largest number of transcript changes; however, salt resulted in mostly a decreased abundance of transcripts, whereas heat shock resulted in mostly an increased abundance of transcripts. A total of 384 transcripts were common to all stress treatments and not expressed in light-grown seedlings; 146 transcripts were present in light-grown seedlings and absent from all stress treatments. A complex pattern of overlapping transcripts between treatments was found, and a significant pattern of congruence in the direction of transcript change between pairs of treatments was uncovered. From the analysis, it appears that the scope of gene expression changes is determined by the challenge, indicating specificity in perception and response. Nonetheless, transcripts regulated by multiple responses are generally affected in the same manner, indicating common or converging regulatory networks. The data are available for additional analysis through a searchable database.

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).

Transcriptomic, proteomic and metabolomic analysis of maize responses to UV-B: Comparison of greenhouse and field growth conditions

UV-B radiation from normal solar fluence elicits physiological and developmental changes in plants under fluctuating environmental conditions. Most UV photobiology studies in plants utilize controlled greenhouse and growth chamber environments in which few conditions vary except the brief presence of UV-B radiation. Our purpose was to compare responses to UV-B in irradiated and shielded maize organs in field (natural solar plus 2x solar supplementation for defined periods) and greenhouse (2x solar supplementation only) conditions during a 4 hour exposure. Three parameters were assessed -- transcripts, proteins, and metabolites -- to determine the degree of overlap in maize responses in field and greenhouse conditions. We assessed irradiated leaves, and both shielded leaves and immature ears. After comparing transcriptome, proteome and metabolome profiles, we find there are more differences than similarities between field and greenhouse responses.

MS23, a master basic helix-loop-helix factor, regulates the specification and development of the tapetum in maize.

Successful male gametogenesis involves orchestration of sequential gene regulation for somatic differentiation in pre-meiotic anthers. We report here the cloning of Male Sterile23 (Ms23), encoding an anther-specific predicted basic helix-loop-helix (bHLH) transcription factor required for tapetal differentiation; transcripts localize initially to the precursor secondary parietal cells then predominantly to daughter tapetal cells. In knockout ms23-ref mutant anthers, five instead of the normal four wall layers are observed. Microarray transcript profiling demonstrates a more severe developmental disruption in ms23-ref than in ms32 anthers, which possess a different bHLH defect. RNA-seq and proteomics data together with yeast two-hybrid assays suggest that MS23 along with MS32, bHLH122 and bHLH51 act sequentially as either homo- or heterodimers to choreograph tapetal development. Among them, MS23 is the earliest-acting factor, upstream of bHLH51 and bHLH122, controlling tapetal specification and maturation. By contrast, MS32 is constitutive and independently regulated and is required later than MS23 in tapetal differentiation. Summary: A cascade of basic helix-loop-helix transcription factors guides tapetal cell development in maize anthers, using proteins conserved in Arabidopsis and rice but deployed with distinctive timing.

UV-B signaling in maize: transcriptomic and metabolomic studies at different irradiation times.

Plants have evolved adaptations to environmental factors, including UV-B present in solar radiation. Deployment of specific adaptive phenotypes to avoid or repair UV-B damage requires physiological and developmental acclimation to variable UV-B fluence. To gain a better understanding of the events in UV-B acclimation, we have analyzed a 5min to 6h time course of transcriptome and metabolome responses in irradiated and shielded leaves and in immature maize ears to unravel the systemic physiological and developmental responses in exposed and shielded organs. Within 10 min of UV-B exposure, transcripts are changed not only in irradiated leaves, but also in shielded tissues. The number of UV-B-regulated transcripts rapidly increases with exposure length. Interestingly, after 10 min of exposure, the overlap in transcriptome changes in irradiated and shielded organs is significant; while, after 6h of UV-B, most transcripts are specific for each tissue under study. We suggest that early events in all tissues may be elicited by common signaling pathways, while at longer exposure times responses become more organ-specific. Our working hypothesis is that mobile signaling molecules are generated in irradiated organs to elicit the initial responses. We found several metabolites that rapidly change after different treatments during the timecourse; myoinositol is one candidate metabolite based on its rapid modulation in all organs. There is also support from RNA profiling: after 1h UV-B, transcripts for myoinositol-1-phosphate synthase are decreased in both irradiated and shielded leaves suggesting downregulation of biogenesis.