Transcriptome response to glyphosate in sensitive and resistant soybean.

The majority of soybeans planted in the United States are resistant to glyphosate due to introduction of a gene encoding for a glyphosate-insensitive 5-enolypyruvylshikimate-3-phosphate synthase. Gene expression profiling was conducted using cDNA microarrays to address questions related to potential secondary effects of glyphosate. When glyphosate-sensitive plants were treated with glyphosate, 3, 170, and 311 genes were identified as having different transcript levels at 1, 4, and 24 h post-treatment (hpt), respectively. Differentially expressed genes were classified into functional categories, and their possible roles in response to glyphosate are briefly discussed. Gene expression profiling of glyphosate-resistant plants treated with glyphosate indicated that the plants were marginally affected at 1 hpt and then quickly adjusted to glyphosate treatment. Ten, four, and four genes were identified as differentially expressed at 1, 4, and 24 hpt. When gene expression profiles of cotyledons from developing seed were compared between the near-isogenic resistant and sensitive lines, two genes were identified as significantly differentially expressed out of 27000, which was less than the empirical false-discovery rate determined from a control experiment. Quantitative real-time reverse-transcribed Polymerase Chain Reaction was conducted on selected genes and yielded results consistent with those from the microarrays. Collectively, these data indicate that there are no major transcriptomic changes associated with currently used glyphosate-resistant soybean.

Sequence-function analysis of the K+-selective family of ion channels using a comprehensive alignment and the KcsA channel structure.

Sequence-function analysis of K(+)-selective channels was carried out in the context of the 3.2 A crystal structure of a K(+) channel (KcsA) from Streptomyces lividans (Doyle et al., 1998). The first step was the construction of an alignment of a comprehensive set of K(+)-selective channel sequences forming the putative permeation path. This pathway consists of two transmembrane segments plus an extracellular linker. Included in the alignment are channels from the eight major classes of K(+)-selective channels from a wide variety of species, displaying varied rectification, gating, and activation properties. Segments of the alignment were assigned to structural motifs based on the KcsA structure. The alignment's accuracy was verified by two observations on these motifs: 1), the most variability is shown in the turret region, which functionally is strongly implicated in susceptibility to toxin binding; and 2), the selectivity filter and pore helix are the most highly conserved regions. This alignment combined with the KcsA structure was used to assess whether clusters of contiguous residues linked by hydrophobic or electrostatic interactions in KcsA are conserved in the K(+)-selective channel family. Analysis of sequence conservation patterns in the alignment suggests that a cluster of conserved residues is critical for determining the degree of K(+) selectivity. The alignment also supports the near-universality of the "glycine hinge" mechanism at the center of the inner helix for opening K channels. This mechanism has been suggested by the recent crystallization of a K channel in the open state. Further, the alignment reveals a second highly conserved glycine near the extracellular end of the inner helix, which may be important in minimizing deformation of the extracellular vestibule as the channel opens. These and other sequence-function relationships found in this analysis suggest that much of the permeation path architecture in KcsA is present in most K(+)-selective channels. Because of this finding, the alignment provides a robust starting point for homology modeling of the permeation paths of other K(+)-selective channel classes and elucidation of sequence-function relationships therein. To assay these applications, a homology model of the Shaker A channel permeation path was constructed using the alignment and KcsA as the template, and its structure evaluated in light of established structural criteria.

Clustering of microarray data reveals transcript patterns associated with somatic embryogenesis in soybean.

Globular somatic embryos can be induced from immature cotyledons of soybean (Glycine max L. Merr. cv Jack) placed on high levels of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos develop from the adaxial side of the cotyledon, whereas the abaxial side evolves into a callus. Using a 9,280-cDNA clone array, we have compared steady-state RNA from the adaxial side from which embryos develop and from the abaxial callus at five time points over the course of the 4 weeks necessary for the development of globular embryos. In a second set of experiments, we have profiled the expression of each clone in the adaxial side during the same period. A total of 495 genes differentially expressed in at least one of these experiments were grouped according to the similarity of their expression profiles using a nonhierarchical clustering algorithm. Our results indicate that the appearance of somatic embryos is preceded by dedifferentiation of the cotyledon during the first 2 weeks on auxin. Changes in mRNA abundance of genes characteristic of oxidative stress and genes indicative of cell division in the adaxial side of the cotyledons suggest that the arrangement of the new cells into organized structures might depend on a genetically controlled balance between cell proliferation and cell death. Our data also suggest that the formation of somatic globular embryos is accompanied by the transcription of storage proteins and the synthesis of gibberellic acid.