Nina V. Fedoroff

Molecular identification and isolation of the Waxy locus in maize

The Waxy (Wx) locus in maize determines the amylose content of pollen and endosperm tissue. There are several mutant alleles of the locus caused by insertion of transposable controlling elements. In the present study, we have used the properties of controlling element alleles to identify the Wx locus and its gene product, with the subsequent objective of isolating the elements causing the mutations. We present evidence that the Wx locus encodes a starch granule-bound 58 kd polypeptide that is synthesized in vitro as a 65 kd precursor. We describe the isolation of recombinant plasmids containing cDNA inserts homologous to Wx mRNA and a recombinant lambda phage containing a genomic Eco RI fragment encompassing most or all of the Wx transcription unit. We show that a mutation caused by the controlling element Dissociation (Ds) is attributable to an insertion of approximately 2.4 kb at the Wx locus.

A Mutation in the Arabidopsis HYL1 Gene Encoding a dsRNA Binding Protein Affects Responses to Abscisic Acid, Auxin, and Cytokinin

Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant’s responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N-(1-naphthyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1, encodes a 419–amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein–protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level.

Radically Rethinking Agriculture for the 21st Century

Population growth, arable land and fresh water limits, and climate change have profound implications for the ability of agriculture to meet this century’s demands for food, feed, fiber, and fuel while reducing the environmental impact of their production. Success depends on the acceptance and use of contemporary molecular techniques, as well as the increasing development of farming systems that use saline water and integrate nutrient flows.

Different Signaling and Cell Death Roles of Heterotrimeric G Protein α and β Subunits in the Arabidopsis Oxidative Stress Response to Ozone

Arabidopsis thaliana plants with null mutations in the genes encoding the α and β subunits of the single heterotrimeric G protein are less and more sensitive, respectively, to O3 damage than wild-type Columbia-0 plants. The first peak of the bimodal oxidative burst elicited by O3 in wild-type plants is almost entirely missing in both mutants. The late peak is normal in plants lacking the Gβ protein but missing in plants lacking the Gα protein. Endogenous reactive oxygen species (ROS) are first detectable in chloroplasts of leaf epidermal guard cells. ROS production in adjacent cells is triggered by extracellular ROS signals produced by guard cell membrane-associated NADPH oxidases encoded by the AtrbohD and AtrbohF genes. The late, tissue damage–associated component of the oxidative burst requires only the Gα protein and arises from multiple cellular sources. The early component of the oxidative burst, arising primarily from chloroplasts, requires signaling through the heterotrimer (or the Gβγ complex) and is separable from Gα-mediated activation of membrane-bound NADPH oxidases necessary for both intercellular signaling and cell death.

Dynamic modeling of gene expression data

We describe the time evolution of gene expression levels by using a time translational matrix to predict future expression levels of genes based on their expression levels at some initial time. We deduce the time translational matrix for previously published DNA microarray gene expression data sets by modeling them within a linear framework by using the characteristic modes obtained by singular value decomposition. The resulting time translation matrix provides a measure of the relationships among the modes and governs their time evolution. We show that a truncated matrix linking just a few modes is a good approximation of the full time translation matrix. This finding suggests that the number of essential connections among the genes is small.

The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1

The results of genetic studies in Arabidopsis indicate that three proteins, the RNase III DICER-Like1 (DCL1), the dsRNA-binding protein HYPONASTIC LEAVES1 (HYL1), and the C2H2 Zn-finger protein SERRATE (SE), are required for the accurate processing of microRNA (miRNA) precursors in the plant cell nucleus. To elucidate the biochemical mechanism of miRNA processing, we developed an in vitro miRNA processing assay using purified recombinant proteins. We find that DCL1 alone releases 21-nt short RNAs from dsRNA as well as synthetic miR167b precursor RNAs. However, correctly processed miRNAs constitute a minority of the cleavage products. We show that recombinant HYL1 and SE proteins accelerate the rate of DCL1-mediated cleavage of pre- and pri-miR167b substrates and promote accurate processing.

The nucleotide sequence of the maize controlling element Activator

We have determined the nucleotide sequence of the transposable maize controlling element Activator (Ac). The Ac element is 4563 bp long and has an imperfect terminal repetition of 11 bp. The element contains two open reading frames (ORF) encoding polypeptides of 839 and 210 amino acids. Evidence derived from structural analysis of a closely related, but transposition-defective Dissociation (Ds) element indicates that the large ORF is the structural gene for a trans-acting function required for transposition. The two ORFs diverge from a short intergenic region which contains characteristic eucaryotic transcription initiation sequences.

Characterizing the stress/defense transcriptome of Arabidopsis

BackgroundTo understand the gene networks that underlie plant stress and defense responses, it is necessary to identify and characterize the genes that respond both initially and as the physiological response to the stress or pathogen develops. We used PCR-based suppression subtractive hybridization to identify Arabidopsis genes that are differentially expressed in response to ozone, bacterial and oomycete pathogens and the signaling molecules salicylic acid (SA) and jasmonic acid.ResultsWe identified a total of 1,058 differentially expressed genes from eight stress cDNA libraries. Digital northern analysis revealed that 55% of the stress-inducible genes are rarely transcribed in unstressed plants and 17% of them were not previously represented in Arabidopsis expressed sequence tag databases. More than two-thirds of the genes in the stress cDNA collection have not been identified in previous studies as stress/defense response genes. Several stress-responsive cis-elements showed a statistically significant over-representation in the promoters of the genes in the stress cDNA collection. These include W- and G-boxes, the SA-inducible element, the abscisic acid response element and the TGA motif.ConclusionsThe stress cDNA collection comprises a broad repertoire of stress-responsive genes encoding proteins that are involved in both the initial and subsequent stages of the physiological response to abiotic stress and pathogens. This set of stress-, pathogen- and hormone-modulated genes is an important resource for understanding the genetic interactions underlying stress signaling and responses and may contribute to the characterization of the stress transcriptome through the construction of standardized specialized arrays.

Arabidopsis primary microRNA processing proteins HYL1 and DCL1 define a nuclear body distinct from the Cajal body

Small regulatory microRNAs (miRNAs) are encoded in long precursors and are released from them during processing by cleavage within partially duplexed stem–loop structures. In the present work we investigated the role of the Arabidopsis nuclear RNA-binding protein HYL1 and the nuclear RNase III enzyme DCL1 in processing of primary miRNA (pri-miR171a). The miR171a gene is complex, with multiple transcription start sites, as well as alternative splicing of exons and alternative polyadenylation sites. Both HYL1 and DCL1 proteins are required for processing of the major pri-miR171a, spliced and polyadenylated forms of which accumulate in plants homozygous for mutations in either gene, but not in wild-type plants. In transiently transfected Arabidopsis protoplasts, HYL1-mCherry and YFP-DCL1 fusion proteins colocalize to small nuclear bodies similar to Cajal bodies but lacking the Cajal body marker Atcoilin. The HYL1 protein coimmunoprecipitates with miR171a and miR159a precursors, indicating that it is an integral component of the precursor processing machinery. Thus, the distinct HYL1- and DCL1-containing nuclear bodies may be miRNA precursor processing sites. Alternatively, they may be assembly and storage sites for the miRNA precursor processing machinery.

Arabidopsis bZIP60 Is a Proteolysis-Activated Transcription Factor Involved in the Endoplasmic Reticulum Stress Response

Proteins synthesized in the endoplasmic reticulum (ER) of eukaryotic cells must be folded correctly before translocation out of the ER. Disruption of protein folding results in the induction of genes for ER-resident chaperones, for example, BiP. This phenomenon is known as the ER stress response. We report here that bZIP60, an Arabidopsis thaliana basic leucine zipper (bZIP) transcription factor with a transmembrane domain, is involved in the ER stress response. When compared with wild-type Arabidopsis plants, homozygous bzip60 mutant plants show a markedly weaker induction of many ER stress-responsive genes. The bZIP60 protein resides in the ER membrane under unstressed condition and is cleaved in response to ER stress caused by either tunicamycin or DTT. The N-terminal fragment containing the bZIP domain is then translocated into the nucleus. Cleavage of bZIP60 is independent of the function of Arabidopsis homologs of mammalian S1P and S2P proteases, which mediate the proteolytic cleavage of the mammalian transcription factor ATF6. In Arabidopsis, expression of the bZIP60 gene and cleavage of the bZIP60 protein are observed in anthers in the absence of stress treatment, suggesting that the ER stress response functions in the normal development of active secretory cells.