Stephen J. Powers

Genetic Characterization and Functional Analysis of the GID1 Gibberellin Receptors in Arabidopsis

We investigated the physiological function of three Arabidopsis thaliana homologs of the gibberellin (GA) receptor GIBBERELLIN-INSENSITIVE DWARF1 (GID1) by determining the developmental consequences of GID1 inactivation in insertion mutants. Although single mutants developed normally, gid1a gid1c and gid1a gid1b displayed reduced stem height and lower male fertility, respectively, indicating some functional specificity. The triple mutant displayed a dwarf phenotype more severe than that of the extreme GA-deficient mutant ga1-3. Flower formation occurred in long days but was delayed, with severe defects in floral organ development. The triple mutant did not respond to applied GA. All three GID1 homologs were expressed in most tissues throughout development but differed in expression level. GA treatment reduced transcript abundance for all three GID1 genes, suggesting feedback regulation. The DELLA protein REPRESSOR OF ga1-3 (RGA) accumulated in the triple mutant, whose phenotype could be partially rescued by loss of RGA function. Yeast two-hybrid and in vitro pull-down assays confirmed that GA enhances the interaction between GID1 and DELLA proteins. In addition, the N-terminal sequence containing the DELLA domain is necessary for GID1 binding. Furthermore, yeast three-hybrid assays showed that the GA-GID1 complex promotes the interaction between RGA and the F-box protein SLY1, a component of the SCFSLY1 E3 ubiquitin ligase that targets the DELLA protein for degradation.

Real-Time Quantitative RT-PCR: Design, Calculations, and Statistics

Two recent letters to the editor of The Plant Cell ([Gutierrez et al., 2008][1]; [Udvardi et al., 2008][2]) highlighted the importance of following correct experimental protocol in quantitative RT-PCR (qRT-PCR). In these letters, the authors outlined measures to allow precise estimation of gene

Inhibition of SNF1-Related Protein Kinase1 Activity and Regulation of Metabolic Pathways by Trehalose-6-Phosphate

Trehalose-6-phosphate (T6P) is a proposed signaling molecule in plants, yet how it signals was not clear. Here, we provide evidence that T6P functions as an inhibitor of SNF1-related protein kinase1 (SnRK1; AKIN10/AKIN11) of the SNF1-related group of protein kinases. T6P, but not other sugars and sugar phosphates, inhibited SnRK1 in Arabidopsis (Arabidopsis thaliana) seedling extracts strongly (50%) at low concentrations (1–20 μm). Inhibition was noncompetitive with respect to ATP. In immunoprecipitation studies using antibodies to AKIN10 and AKIN11, SnRK1 catalytic activity and T6P inhibition were physically separable, with T6P inhibition of SnRK1 dependent on an intermediary factor. In subsequent analysis, T6P inhibited SnRK1 in extracts of all tissues analyzed except those of mature leaves, which did not contain the intermediary factor. To assess the impact of T6P inhibition of SnRK1 in vivo, gene expression was determined in seedlings expressing Escherichia coli otsA encoding T6P synthase to elevate T6P or otsB encoding T6P phosphatase to decrease T6P. SnRK1 target genes showed opposite regulation, consistent with the regulation of SnRK1 by T6P in vivo. Analysis of microarray data showed up-regulation by T6P of genes involved in biosynthetic reactions, such as genes for amino acid, protein, and nucleotide synthesis, the tricarboxylic acid cycle, and mitochondrial electron transport, which are normally down-regulated by SnRK1. In contrast, genes involved in photosynthesis and degradation processes, which are normally up-regulated by SnRK1, were down-regulated by T6P. These experiments provide strong evidence that T6P inhibits SnRK1 to activate biosynthetic processes in growing tissues.

Quantitative resistance increases the durability of qualitative resistance to Leptosphaeria maculans in Brassica napus.

It has frequently been hypothesized that quantitative resistance increases the durability of qualitative (R-gene mediated) resistance but supporting experimental evidence is rare. To test this hypothesis, near-isogenic lines with/without the R-gene Rlm6 introduced into two Brassica napus cultivars differing in quantitative resistance to Leptosphaeria maculans were used in a 5-yr field experiment. Recurrent selection of natural fungal populations was done annually on each of the four plant genotypes, using crop residues from each genotype to inoculate separately the four series of field trials for five consecutive cropping seasons. Severity of phoma stem canker was measured on each genotype and frequencies of avirulence alleles in L. maculans populations were estimated. Recurrent selection of virulent isolates by Rlm6 in a susceptible background rendered the resistance ineffective by the third cropping season. By contrast, the resistance was still effective after 5 yr of selection by the genotype combining this gene with quantitative resistance. No significant variation in the performance of quantitative resistance alone was noted over the course of the experiment. We conclude that quantitative resistance can increase the durability of Rlm6. We recommend combining quantitative resistance with R-gene mediated resistance to enhance disease control and crop production.

Genetic Analysis Reveals That C19-GA 2-Oxidation Is a Major Gibberellin Inactivation Pathway in Arabidopsis

Bioactive hormone concentrations are regulated both at the level of hormone synthesis and through controlled inactivation. Based on the ubiquitous presence of 2β-hydroxylated gibberellins (GAs), a major inactivating pathway for the plant hormone GA seems to be via GA 2-oxidation. In this study, we used various approaches to determine the role of C19-GA 2-oxidation in regulating GA concentration and GA-responsive plant growth and development. We show that Arabidopsis thaliana has five C19-GA 2-oxidases, transcripts for one or more of which are present in all organs and at all stages of development examined. Expression of four of the five genes is subject to feed-forward regulation. By knocking out all five Arabidopsis C19-GA 2-oxidases, we show that C19-GA 2-oxidation limits bioactive GA content and regulates plant development at various stages during the plant life cycle: C19-GA 2-oxidases prevent seed germination in the absence of light and cold stimuli, delay the vegetative and floral phase transitions, limit the number of flowers produced per inflorescence, and suppress elongation of the pistil prior to fertilization. Under GA-limited conditions, further roles are revealed, such as limiting elongation of the main stem and side shoots. We conclude that C19-GA 2-oxidation is a major GA inactivation pathway regulating development in Arabidopsis.

Identification of Human-Derived Volatile Chemicals that Interfere with Attraction of Aedes aegypti Mosquitoes

It is known that human individuals show different levels of attractiveness to mosquitoes. In this study, we investigated the chemical basis for low attractiveness. We recorded behaviors of Aedes aegypti toward the hands of human volunteers and toward the volatile chemicals produced by their bodies. Some individuals, and their corresponding volatiles, elicited low upwind flight, relative attraction, and probing activity. Analyzing the components by gas chromatography coupled to electrophysiological recordings from the antennae of Aedes aegypti, enabled the location of 33 physiologically relevant compounds. The results indicated that higher levels of specific compounds may be responsible for decreased “attractiveness.” In behavioral experiments, five of the compounds caused a significant reduction in upwind flight of Aedes aegypti to attractive human hands. Thus, unattractiveness of individuals may result from a repellent, or attractant “masking,” mechanism.

Transcriptome and Metabolite Profiling of the Infection Cycle of Zymoseptoria tritici on Wheat Reveals a Biphasic Interaction with Plant Immunity Involving Differential Pathogen Chromosomal Contributions and a Variation on the Hemibiotrophic Lifestyle Definition

The temporal dynamics of Zymoseptoria tritici reproduction on Triticum aestivum involves a biphasic manipulation of plant defense responses. The hemibiotrophic fungus Zymoseptoria tritici causes Septoria tritici blotch disease of wheat (Triticum aestivum). Pathogen reproduction on wheat occurs without cell penetration, suggesting that dynamic and intimate intercellular communication occurs between fungus and plant throughout the disease cycle. We used deep RNA sequencing and metabolomics to investigate the physiology of plant and pathogen throughout an asexual reproductive cycle of Z. tritici on wheat leaves. Over 3,000 pathogen genes, more than 7,000 wheat genes, and more than 300 metabolites were differentially regulated. Intriguingly, individual fungal chromosomes contributed unequally to the overall gene expression changes. Early transcriptional down-regulation of putative host defense genes was detected in inoculated leaves. There was little evidence for fungal nutrient acquisition from the plant throughout symptomless colonization by Z. tritici, which may instead be utilizing lipid and fatty acid stores for growth. However, the fungus then subsequently manipulated specific plant carbohydrates, including fructan metabolites, during the switch to necrotrophic growth and reproduction. This switch coincided with increased expression of jasmonic acid biosynthesis genes and large-scale activation of other plant defense responses. Fungal genes encoding putative secondary metabolite clusters and secreted effector proteins were identified with distinct infection phase-specific expression patterns, although functional analysis suggested that many have overlapping/redundant functions in virulence. The pathogenic lifestyle of Z. tritici on wheat revealed through this study, involving initial defense suppression by a slow-growing extracellular and nutritionally limited pathogen followed by defense (hyper) activation during reproduction, reveals a subtle modification of the conceptual definition of hemibiotrophic plant infection.

Analysis of the Developmental Roles of the Arabidopsis Gibberellin 20-Oxidases Demonstrates That GA20ox1, -2, and -3 Are the Dominant Paralogs

In a systematic study of the five Arabidopsis thaliana genes that encode the biosynthetic enzyme gibberellin 20-oxidase, we show by mutant analysis and examination of gene expression that three of the genes make the major contribution to plant height and fertility, with the other two genes playing minor roles. Gibberellin (GA) biosynthesis is necessary for normal plant development, with later GA biosynthetic stages being governed by multigene families. Arabidopsis thaliana contains five GA 20-oxidase (GA20ox) genes, and past work has demonstrated the importance of GA20ox1 and -2 for growth and fertility. Here, we show through systematic mutant analysis that GA20ox1, -2, and -3 are the dominant paralogs; their absence results in severe dwarfism and almost complete loss of fertility. In vitro analysis revealed that GA20ox4 has full GA20ox activity, but GA20ox5 catalyzes only the first two reactions of the sequence by which GA12 is converted to GA9. GA20ox3 functions almost entirely redundantly with GA20ox1 and -2 at most developmental stages, including the floral transition, while GA20ox4 and -5 have very minor roles. These results are supported by analysis of the gene expression patterns in promoter:β-glucuronidase reporter lines. We demonstrate that fertility is highly sensitive to GA concentration, that GA20ox1, -2, and -3 have significant effects on floral organ growth and anther development, and that both GA deficiency and overdose impact on fertility. Loss of GA20ox activity causes anther developmental arrest, with the tapetum failing to degrade. Some phenotypic recovery of late flowers in GA-deficient mutants, including ga1-3, indicated the involvement of non-GA pathways in floral development.

Inhibition of SnRK1 by metabolites: Tissue-dependent effects and cooperative inhibition by glucose 1-phosphate in combination with trehalose 6-phosphate

SnRK1 of the SNF1/AMPK group of protein kinases is an important regulatory protein kinase in plants. SnRK1 was recently shown as a target of the sugar signal, trehalose 6-phosphate (T6P). Glucose 6-phosphate (G6P) can also inhibit SnRK1 and given the similarity in structure to T6P, we sought to establish if each could impart distinct inhibition of SnRK1. Other central metabolites, glucose 1-phosphate (G1P), fructose 6-phosphate and UDP-glucose were also tested, and additionally ribose 5-phosphate (R5P), recently reported to inhibit SnRK1 strongly in wheat grain tissue. For the metabolites that inhibited SnRK1, kinetic models show that T6P, G1P and G6P each provide distinct regulation (50% inhibition of SnRK1 at 5.4 μM, 480 μM, >1 mM, respectively). Strikingly, G1P in combination with T6P inhibited SnRK1 synergistically. R5P, in contrast to the other inhibitors, inhibited SnRK1 in green tissues only. We show that this is due to consumption of ATP in the assay mediated by phosphoribulokinase during conversion of R5P to ribulose-1,5-bisphosphate. The accompanying loss of ATP limits the activity of SnRK1 giving rise to an apparent inhibition of SnRK1. Inhibition of SnRK1 by R5P in wheat grain preparations can be explained by the presence of green pericarp tissue; this exposes an important caveat in the assessment of potential protein kinase inhibitors. Data provide further insight into the regulation of SnRK1 by metabolites.

GCN2-dependent phosphorylation of eukaryotic translation initiation factor-2α in Arabidopsis

The yeast regulatory protein kinase, general control non-derepressible-2 (GCN2) plays a key role in general amino acid control. GCN2 phosphorylates the α subunit of the trimeric eukaryotic translation initiation factor-2 (eIF2), bringing about a decrease in the general rate of protein synthesis but an increase in the synthesis of GCN4, a transcription factor that promotes the expression of genes encoding enzymes for amino acid biosynthesis. The present study concerned the phosphorylation of Arabidopsis eIF2α (AteIF2α) by the Arabidopsis homologue of GCN2, AtGCN2, and the role of AtGCN2 in regulating genes encoding enzymes of amino acid biosynthesis and responding to virus infection. A null mutant for AtGCN2 called GT8359 was obtained and western analysis confirmed that it lacked AtGCN2 protein. GT8359 was more sensitive than wild-type Arabidopsis to herbicides that affect amino acid biosynthesis. Phosphorylation of AteIF2α occurred in response to herbicide treatment but only in wild-type Arabidopsis, not GT8359, showing it to be AtGCN2-dependent. Expression analysis of genes encoding key enzymes for amino acid biosynthesis and nitrate assimilation revealed little effect of loss of AtGCN2 function in GT8359 except that expression of a nitrate reductase gene, NIA1, was decreased. Analysis of wild-type and GT8359 plants infected with Turnip yellow mosaic virus or Turnip crinkle virus showed that AteIF2α was not phosphorylated.