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Virginia Walbot

Publications and source records attributed to Virginia Walbot.

At least 19 recordsLinked to original sources
PubMed2006

Comparative profiling of the sense and antisense transcriptome of maize lines.

BACKGROUND: There 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. RESULTS: Four 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. CONCLUSION: Despite 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.

Expressed Sequence Tags↗
PubMed2006

Genome-wide analysis of high-altitude maize and gene knockdown stocks implicates chromatin remodeling proteins in response to UV-B.

A comparative analysis, by expression profiling of maize, was performed to identify novel components in the mechanisms of maize responses to UV-B. Five high-altitude landraces grown from 2,000 to 3,400 m naturally receive higher UV-B fluence than plants at lower altitudes and similar latitudes. These high-altitude landraces were compared directly with a low-altitude line and with literature reports for other temperate maize lines. A microarray analysis demonstrated that among the UV-B responsive transcripts, several types of gene implicated in chromatin remodeling are differentially expressed before and after UV-B treatment in high-altitude lines. RNAi transgenic plants with lower expression of four such chromatin-associated genes exhibited hypersensitivity to UV-B by measurements of leaf arching, increased leaf chlorosis and necrosis, and altered UV-B regulation of selected genes. These results collectively suggest that genes involved in chromatin remodeling are crucial for UV-B acclimation and that some high-altitude lines exhibit adaptations to this challenge.

Adaptation, Physiological↗
PubMed2005

Analysis of leaf proteome after UV-B irradiation in maize lines differing in sensitivity.

UV-B radiation causes diverse morphological and physiological responses in plants, but the underlying mechanisms governing these integrated responses are unknown. In this study, we systematically surveyed responses of maize leaves to UV-B radiation using DIGE 2D gels and identified selected proteins by mass spectrometry and immunodetection analysis. To identify changes in protein accumulation in response to UV-B radiation, a line (b, pl W23) deficient in flavonoid sunscreen compounds and hence similar to commercial corn was used. In addition, its proteome in natural UV-B conditions was compared with that of two maize landraces from high altitudes (Cacahuacintle and Confite Puneño) that have improved UV-B tolerance. Protein patterns in adult maize leaves (Zea mays) were documented after growth for 21 days in sunlight depleted of UV-B radiation or growth in sunlight including an 8-h UV-B supplementation during 1 day in the field. We found that there is a very high correlation between previously documented mRNA accumulation assessed by microarray hybridization and quantitative real time reverse transcription-PCR and protein expression after UV-B irradiation in leaves of W23. Multiple isoforms were confirmed for some proteins; at least one protein, pyruvate, phosphate dikinase, is regulated post-translationally by phosphorylation by UV-B exposure. Proteins differentially regulated by UV-B radiation in W23 with higher levels under similar UV-B conditions in high altitude plants were also identified. These could be genetically fixed traits conferring UV-B tolerance and offer clues to specific adaptations to living in high ambient UV-B conditions.

Amino Acid Sequence↗
PubMed2004

Crosslinking of ribosomal proteins to RNA in maize ribosomes by UV-B and its effects on translation.

Ultraviolet-B (UV-B) photons can cause substantial cellular damage in biomolecules, as is well established for DNA. Because RNA has the same absorption spectrum for UV as DNA, we have investigated damage to this cellular constituent. In maize (Zea mays) leaves, UV-B radiation damages ribosomes by crosslinking cytosolic ribosomal proteins S14, L23a, and L32, and chloroplast ribosomal protein L29 to RNA. Ribosomal damage accumulated during a day of UV-B exposure correlated with a progressive decrease in new protein production; however, de novo synthesis of some ribosomal proteins is increased after 6 h of UV-B exposure. After 16 h without UV-B, damaged ribosomes were eliminated and translation was restored to normal levels. Ribosomal protein S6 and an S6 kinase are phosphorylated during UV-B exposure; these modifications are associated with selective translation of some ribosomal proteins after ribosome damage in mammalian fibroblast cells and may be an adaptation in maize. Neither photosynthesis nor pigment levels were affected significantly by UV-B, demonstrating that the treatment applied is not lethal and that maize leaf physiology readily recovers.

Dose-Response Relationship, Radiation↗
PubMed2004

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.

DNA Mutational Analysis↗
PubMed2004

A multidrug resistance-associated protein involved in anthocyanin transport in Zea mays.

Anthocyanin biosynthesis is one of the most thoroughly studied enzymatic pathways in biology, but little is known about the molecular mechanisms of its final stage: the transport of the anthocyanin pigment into the vacuole. We have identified a multidrug resistance-associated protein (MRP), ZmMrp3, that is required for this transport process in maize (Zea mays). ZmMrp3 expression is controlled by the regulators of anthocyanin biosynthesis and mirrors the expression of other anthocyanin structural genes. Localization of ZmMRP3 in vivo shows its presence in the tonoplast, the site at which anthocyanin transport occurs. Mutants generated using antisense constructs have a distinct pigmentation phenotype in the adult plant that results from a mislocalization of the pigment as well as significant reduction in anthocyanin content, with no alteration in the anthocyanin species produced. Surprisingly, mutant plants did not show a phenotype in the aleurone. This appears to reflect the presence of a second, highly homologous gene, ZmMrp4, that is also coregulated with the anthocyanin pathway but is expressed exclusively in aleurone tissue. This description of a plant MRP with a role in the transport of a known endogenous substrate provides a new model system for examining the biological and biochemical mechanisms involved in the MRP-mediated transport of plant secondary metabolites.

Amino Acid Sequence↗
PubMed2004

Rapid transcriptome responses of maize (Zea mays) to UV-B in irradiated and shielded tissues.

BACKGROUND: Depletion of stratospheric ozone has raised terrestrial levels of ultraviolet-B radiation (UV-B), an environmental change linked to an increased risk of skin cancer and with potentially deleterious consequences for plants. To better understand the processes of UV-B acclimation that result in altered plant morphology and physiology, we investigated gene expression in different organs of maize at several UV-B fluence rates and exposure times. RESULTS: Microarray hybridization was used to assess UV-B responses in directly exposed maize organs and organs shielded by a plastic that absorbs UV-B. After 8 hours of high UV-B, the abundance of 347 transcripts was altered: 285 were increased significantly in at least one organ and 80 were downregulated. More transcript changes occurred in directly exposed than in shielded organs, and the levels of more transcripts were changed in adult compared to seedling tissues. The time course of transcript abundance changes indicated that the response kinetics to UV-B is very rapid, as some transcript levels were altered within 1 hour of exposure. CONCLUSIONS: Most of the UV-B regulated genes are organ-specific. Because shielded tissues, including roots, immature ears, and leaves, displayed altered transcriptome profiles after exposure of the plant to UV-B, some signal(s) must be transmitted from irradiated to shielded tissues. These results indicate that there are integrated responses to UV-B radiation above normal levels. As the same total UV-B irradiation dose applied at three intensities elicited different transcript profiles, the transcriptome changes exhibit threshold effects rather than a reciprocal dose-effect response. Transcriptome profiling highlights possible signaling pathways and molecules for future research.

Acclimatization↗
PubMed2003

Deletion derivatives of the MuDR regulatory transposon of maize encode antisense transcripts but are not dominant-negative regulators of mutator activities.

The maize MuDR/Mu transposable elements are highly aggressive, and their activities are held in check by host developmental and epigenetic mechanisms. The Mutator regulatory element, MuDR, produces both sense and antisense transcripts. We have investigated the impact of the presence of antisense transcripts on the abundance of the corresponding sense messages and on the regulation of Mutator activities. We report that internal deletions in MuDR arise frequently in somatic tissues; preferential loss of the 3' untranslated region of mudrA and/or mudrB containing the intergenic region is correlated with chimeric sense mudrA/antisense mudrB and sense mudrB/antisense mudrA transcripts. Heritable internal deletions are extremely frequent (>10(-2) per element), and the resulting defective MuDR elements also encode antisense transcripts. Expression of endogenous or additional transgene-encoded antisense transcripts neither decreases sense transcript levels nor inhibits Mutator excision activity over the three generations examined. We propose that antisense transcripts produced by MuDR deletions are not dominant-negative regulators of Mutator activities.

Base Sequence↗
PubMed2003

ZmDB, an integrated database for maize genome research.

Zea mays DataBase (ZmDB) seeks to provide a comprehensive view of maize (corn) genetics by linking genomic sequence data with gene expression analysis and phenotypes of mutant plants. ZmDB originated in 1999 as the Web portal for a large project of maize gene discovery, sequencing and phenotypic analysis using a transposon tagging strategy and expressed sequence tag (EST) sequencing. Recently, ZmDB has broadened its scope to include all public maize ESTs, genome survey sequences (GSSs), and protein sequences. More than 170 000 ESTs are currently clustered into approximately 20 000 contigs and about an equal number of apparent singlets. These clusters are continuously updated and annotated with respect to potential encoded protein products. More than 100 000 GSSs are similarly assembled and annotated by spliced alignment with EST and protein sequences. The ZmDB interface provides quick access to analytical tools for further sequence analysis. Every sequence record is linked to several display options and similarity search tools, including services for multiple sequence alignment, protein domain determination and spliced alignment. Furthermore, ZmDB provides web-based ordering of materials generated in the project, including ESTs, ordered collections of genomic sequences tagged with the RescueMu transposon and microarrays of amplified ESTs. ZmDB can be accessed at http://zmdb.iastate.edu/.

DNA Transposable Elements↗
PubMed2003

Post-transcriptional regulation of expression of the Bronze2 gene of Zea mays L.

The glutathione S-transferase encoded by Bronze2 performs the last genetically defined step in maize anthocyanin biosynthesis, being required for pigment sequestration into vacuoles. The Bz2 primary transcript contains a single intron; in maize leaves both spliced and unspliced Bz2 transcripts are usually present and are predicted to encode 26 and 14 kDa proteins, respectively. To increase understanding of the role and regulation of Bz2 transcript splicing, we examined Bz2 expression during development in transgenic maize plants expressing a 35S:Bz2 (35S:mycBz2i) gene and, by transient expression analysis, in Black Mexican Sweet maize protoplasts. We show here that the gene is expressed in diverse tissues that lack functional copies of one or both transcription factors regulating anthocyanin synthesis, that transcript levels are much higher when the R/B plus C1/Pl transcription factors are present, and that the splicing decision depends on local sequence context. The predicted 14 kDa protein was never detected indicating that unspliced transcripts are likely to be non-coding. The native 26 kDa BZ2 protein is loosely membrane-bound, but it was detectable only in tissues accumulating anthocyanin. Consequently, BZ2 accumulation appears to be limited by stringent post-transcriptional regulation.

5' Untranslated Regions↗
PubMed2003

Unique features of the plant life cycle and their consequences.

Continuous development, the absence of a germline, flexible and reversible cellular differentiation, and the existence of haploid and diploid generations--both of which express genes--are characteristics that distinguish plants from animals. Because these differences alter the impact of mutations, animals and plants experience varied selection pressures. Despite different life-cycles, both flowering plants and multicellular animals have evolved complex sensing mechanisms that act after fertilization as 'quality checks' on reproduction, and that detect chromosome dosage and the parent of origin for specific genes. Although flowering plant embryos escape such surveillance in vitro, embryo success in the seed often depends on a healthy endosperm--a nutritive tissue that is produced by a second fertilization event in which maternal and paternal gene contributions can be monitored immediately after fertilization and throughout development.

Animals↗
PubMed2003

Initiation of silencing of maize MuDR/Mu transposable elements.

Homology-dependent gene silencing contributes to genomic stability through suppression of transposable elements. Co-ordinate epigenetic silencing is the main regulatory mechanism controlling dispersed, multicopy MuDR/Mu elements responsible for Mutator activity in maize. Silencing eliminates transposition and proceeds through transcriptional inactivation of MuDR genes and DNA methylation of the terminal inverted repeats (TIRs) in both the regulatory MuDR and non-autonomous Mu elements. In plants with active MuDR/Mu elements, initiation of silencing coincides with nuclear retention of non-polyadenylated RNA derived from MuDR and recently described MuDR homologs (hMuDR elements). Nuclear accumulation of MuDR/hMuDR RNA is developmentally progressive, paralleling loss of Mutator activity and is predictive of loss of Mu somatic excision in the progeny. A high ratio of nuclear to cytoplasmic RNA is the earliest molecular marker for MuDR silencing suggesting that the nuclear RNA may trigger transcriptional silencing. We also demonstrate the constitutive presence of small transposon-specific RNAs of 21-26 nucleotides in all maize lines tested, independent of the Mutator activity. The role of the small RNAs in transposon silencing and translational regulation of transposon-encoded proteins is discussed.

Cell Nucleus↗
PubMed2003

Gene expression profiling in response to ultraviolet radiation in maize genotypes with varying flavonoid content.

Microarray hybridization was used to assess acclimation responses to four UV regimes by near isogenic maize (Zea mays) lines varying in flavonoid content. We found that 355 of the 2,500 cDNAs tested were regulated by UV radiation in at least one genotype. Among these, 232 transcripts are assigned putative functions, whereas 123 encode unknown proteins. UV-B increased expression of stress response and ribosomal protein genes, whereas photosynthesis-associated genes were down-regulated; lines lacking UV-absorbing pigments had more dramatic responses than did lines with these pigments, confirming the shielding role of these compounds. Sunlight filtered to remove UV-B or UV-B plus UV-A resulted in significant expression changes in many genes not previously associated with UV responses. Some pathways regulated by UV radiation are shared with defense, salt, and oxidative stresses; however, UV-B radiation can activate additional pathways not shared with other stresses.

Flavonoids↗
PubMed2002

Progress in maize gene discovery: a project update.

The Maize Gene Discovery Project (MGDP) is a 5-year NSF-funded plant genome initiative that began in 1998. The MGDP collaboration involves researchers at six universities from diverse disciplines with the common goal of discovering new maize genes and developing tools for the phenotypic characterization of maize mutants. The project utilizes several approaches: EST sequencing, cDNA microarray production, and the discovery of gene function and genomic sequence through the use of a recombinant Mu1 transposon ( RescueMu). Current achievements of the MGDP (NSF 98-72657) include the sequencing of over 120,000 maize ESTs from diverse cDNA libraries, and over 70,000 RescueMu flanking sequences, as well as the cataloguing of mutant seed and cob phenotypes of 23,000 maize ears, 6,200 families of maize seedlings, and 4,000 families of adult maize plants carrying MuDR/Mu and RescueMu insertion alleles. A consolidation of over 24,000 unique sequences from 19 libraries has been made into the first two of the planned set of four "Unigene" microarray slides. In addition, slides for four EST libraries have been produced. These microarray slides, EST clones, library plates of immortalized RescueMu bacterial cultures, and seed are all available online (http://www.zmdb.iastate.edu). The ZmDB website posts periodic assemblies of all maize EST and genomic sequences available from GenBank. ZmDB is also a portal for sequence analysis software designed to aid in gene discovery: MuSeqBox, GeneSeqer, and SplicePredictor. In addition, ZmDB contains links to other plant and genetics websites.

Alleles↗
PubMed2002

Gene-expression profile comparisons distinguish seven organs of maize.

BACKGROUND: A maize array was fabricated with 5,376 unique expressed sequence tag (EST) clones sequenced from 4-day-old roots, immature ears and adult organ cDNA libraries. To elucidate organ relationships, relative mRNA levels were quantified by hybridization with embryos, three maize vegetative organs (leaf blades, leaf sheaths and roots) from multiple developmental stages, husk leaves and two types of floral organs (immature ears and silks). RESULTS: Clustering analyses of the hybridization data suggest that maize utilizes both the PEPCK and NADP-ME C(4) photosynthetic routes as genes in these pathways are co-regulated. Husk RNA has a gene-expression profile more similar to floral organs than to vegetative leaves. Only 7% of the genes were highly organ specific, showing over a fourfold difference in at least one of 12 comparisons and 37% showed a two- to fourfold difference. The majority of genes were expressed in diverse organs with little difference in transcript levels. Cross-hybridization among closely related genes within multigene families could obscure tissue specificity. As a first step in elucidating individual gene-expression patterns, we show that 45-nucleotide oligo probes produce signal intensities and signal ratios comparable to PCR probes on the same matrix. CONCLUSIONS: Gene-expression profile studies with cDNA microarrays provide a new molecular tool for defining plant organs and their relationships and for discovering new biological processes in silico. cDNA microarrays are insufficient for differentiating recently duplicated genes. Gene-specific oligo probes printed along with cDNA probes can query individual gene-expression profiles and gene families simultaneously.

Cluster Analysis↗
PubMed2002

Comparative genomics of Arabidopsis and maize: prospects and limitations.

The completed Arabidopsis genome seems to be of limited value as a model for maize genomics. In addition to the expansion of repetitive sequences in maize and the lack of genomic micro-colinearity, maize-specific or highly-diverged proteins contribute to a predicted maize proteome of about 50,000 proteins, twice the size of that of Arabidopsis.

Arabidopsis↗
PubMed2002

Subcellular localization of MURA and MURB proteins encoded by the maize MuDR transposon.

MuDR controls transposition of the Mu transposable element family in Zea mays L. It produces two major transcripts: mudrA and mudrB, mudrA encodes the MURA transposase, but no specific function has been ascribed to mudrB, which lacks strong homology to known genes. Using transient expression assays in onion epidermal cells, we defined three monopartite nuclear localization signals (NLSs) of MURA; each was functionally sufficient for nuclear targeting of MURA:GUS fusion proteins. Interestingly, one NLS (NLS-A3) is produced by the splicing of the third intron. In contrast, there were no clear NLS in MURB, and the major form of MURB aggregated in the cytoplasm. Self-interaction of MURA and of MURB was also shown in a yeast two-hybrid assay. To test whether interactions of MURA and MURB can occur at the level of protein translocation into the nucleus, a cytoplasmically localized MURB:GFP was co-expressed with MURA or with the GUS fusion proteins. Co-expression did not change the localization pattern of either MURA or MURB; MURA and MURB do not detectably interact in a yeast two-hybrid assay. These results suggest that MURA and MURB do not mutually affect their localization, at least in the forms examined here.

Amino Acid Sequence↗