Co-expression tools for plant biology: opportunities for hypothesis generation and caveats

Gene co‐expression analysis has emerged in the past 5 years as a powerful tool for gene function prediction. In essence, co‐expression analysis asks the question ‘what are the genes that are co‐expressed, that is, those that show similar expression profiles across many experiments, with my gene of interest?’. Genes that are highly co‐expressed may be involved in the biological process or processes of the query gene. This review describes the tools that are available for performing such analyses, how each of these perform, and also discusses statistical issues including how normalization of gene expression data can influence co‐expression results, calculation of co‐expression scores and P values, and the influence of data sets used for co‐expression analysis. Finally, examples from the literature will be presented, wherein co‐expression has been used to corroborate and discover various aspects of plant biology.     

Xeml Lab: a tool that supports the design of experiments at a graphical interface and generates computer-readable metadata files, which capture information about genotypes, growth conditions, environmental perturbations and sampling strategy

Data mining depends on the ability to access machine-readable metadata that describe genotypes, environmental conditions, and sampling times and strategy. This article presents Xeml Lab. The Xeml Interactive Designer provides an interactive graphical interface at which complex experiments can be designed, and concomitantly generates machine-readable metadata files. It uses a new eXtensible Mark-up Language (XML)-derived dialect termed XEML. Xeml Lab includes a new ontology for environmental conditions, called Xeml Environment Ontology. However, to provide versatility, it is designed to be generic and also accepts other commonly used ontology formats, including OBO and OWL. A review summarizing important environmental conditions that need to be controlled, monitored and captured as metadata is posted in a Wiki ( http://www.codeplex.com/XeO ) to promote community discussion. The usefulness of Xeml Lab is illustrated by two meta-analyses of a large set of experiments that were performed with Arabidopsis thaliana during 5 years. The first reveals sources of noise that affect measurements of metabolite levels and enzyme activities. The second shows that Arabidopsis maintains remarkably stable levels of sugars and amino acids across a wide range of photoperiod treatments, and that adjustment of starch turnover and the leaf protein content contribute to this metabolic homeostasis.     

Carboxy Terminal Tail of Polycystin-1 Regulates Localization of TSC2 to Repress mTOR

Autosomal dominant polycystic kidney disease (ADPKD) is a commonly inherited renal disorder caused by defects in the PKD1 or PKD2 genes. ADPKD is associated with significant morbidity, and is a major underlying cause of end-stage renal failure (ESRF). Commonly, treatment options are limited to the management of hypertension, cardiovascular risk factors, dialysis, and transplantation when ESRF develops, although several new pharmacotherapies, including rapamycin, have shown early promise in animal and human studies. Evidence implicates polycystin-1 (PC-1), the gene product of the PKD1 gene, in regulation of the mTOR pathway. Here we demonstrate a mechanism by which the intracellular, carboxy-terminal tail of polycystin-1 (CP1) regulates mTOR signaling by altering the subcellular localization of the tuberous sclerosis complex 2 (TSC2) tumor suppressor, a gatekeeper for mTOR activity. Phosphorylation of TSC2 at S939 by AKT causes partitioning of TSC2 away from the membrane, its GAP target Rheb, and its activating partner TSC1 to the cytosol via 14-3-3 protein binding. We found that TSC2 and a C-terminal polycystin-1 peptide (CP1) directly interact and that a membrane-tethered CP1 protects TSC2 from AKT phosphorylation at S939, retaining TSC2 at the membrane to inhibit the mTOR pathway. CP1 decreased binding of 14-3-3 proteins to TSC2 and increased the interaction between TSC2 and its activating partner TSC1. Interestingly, while membrane tethering of CP1 was required to activate TSC2 and repress mTOR, the ability of CP1 to inhibit mTOR signaling did not require primary cilia and was independent of AMPK activation. These data identify a unique mechanism for modulation of TSC2 repression of mTOR signaling via membrane retention of this tumor suppressor, and identify PC-1 as a regulator of this downstream component of the PI3K signaling cascade.     

Decadal vegetation changes in a northern peatland, greenhouse gas fluxes and net radiative forcing

Thawing permafrost in the sub‐Arctic has implications for the physical stability and biological dynamics of peatland ecosystems. This study provides an analysis of how permafrost thawing and subsequent vegetation changes in a sub‐Arctic Swedish mire have changed the net exchange of greenhouse gases, carbon dioxide (CO₂) and CH₄ over the past three decades. Images of the mire (ca. 17 ha) and surroundings taken with film sensitive in the visible and the near infrared portion of the spectrum, [i.e. colour infrared (CIR) aerial photographs from 1970 and 2000] were used. The results show that during this period the area covered by hummock vegetation decreased by more than 11% and became replaced by wet‐growing plant communities. The overall net uptake of C in the vegetation and the release of C by heterotrophic respiration might have increased resulting in increases in both the growing season atmospheric CO₂ sink function with about 16% and the CH₄ emissions with 22%. Calculating the flux as CO₂ equivalents show that the mire in 2000 has a 47% greater radiative forcing on the atmosphere using a 100‐year time horizon. Northern peatlands in areas with thawing sporadic or discontinuous permafrost are likely to act as larger greenhouse gas sources over the growing season today than a few decades ago because of increased CH₄ emissions.     

Troponin I binds polycystin-L and inhibits its calcium-induced channel activation

Polycystin-L (PCL) is an isoform of polycystin-2, the product of the second gene associated with autosomal dominant polycystic kidney disease, and functions as a Ca(2+)-regulated nonselective cation channel. We recently demonstrated that polycystin-2 interacts with troponin I, an important regulatory component of the actin microfilament complex in striated muscle cells and an angiogenesis inhibitor. In this study, using the two-microelectrode voltage-clamp technique and Xenopus oocyte expression system, we showed that the calcium-induced PCL channel activation is substantially inhibited by the skeletal and cardiac troponin I (60% and 31% reduction, respectively). Reciprocal co-immunoprecipitation experiments demonstrated that PCL physically associates with the skeletal and cardiac troponin I isoforms in overexpressed Xenopus oocytes and mouse fibroblast NIH 3T3 cells. Furthermore, both native PCL and cardiac troponin I were present in human heart tissues where they indeed associate with each other. GST pull-down and microtiter binding assays showed that the C-terminus of PCL interacts with the troponin I proteins. The yeast two-hybrid assay further verified this interaction and defined the corresponding interacting domains of the PCL C-terminus and troponin I. Taken together, this study suggests that troponin I acts as a regulatory subunit of the PCL channel complex and provides the first direct evidence that PCL is associated with the actin cytoskeleton through troponin I.     

Translocation of 32P between interacting mycelia of a wood-decomposing fungus and ectomycorrhizal fungi in microcosm systems

Interactions between saprotrophic and ectomycorrhizal fungi have been largely ignored, although their mycelia often share the same microsites. The mycelial systems show general similarity to each other and, although the enzymatic potential of the saprotrophic fungi is generally considered to be higher, the importance of organic nutrient sources to ectomycorrhizal fungi is now widely accepted. In the experiments described here, nutritional interactions involving transfer of elements from one mycelium to the other have been monitored dynamically using radioactive tracers and a non-destructive electronic autoradiography system. Microcosms were used in which mycelial systems of the ectomycorrhizal fungi Suillus variegatus and Paxillus involutus , extending from Pinus sylvestris host plants, were confronted with mycelia of the saprotroph Hypholoma fasciculare extending from wood blocks. The fungi showed a clear morphological confrontation response. The mycorrhizal mycelium often formed dense patches over the Hypholoma mycelia. Up to 25% of the ³²P present in the Hypholoma mycelium was captured by the mycorrhizal fungi and translocated to the plant host within 30 d. The transfer of ³²P to the saprotroph from labelled mycorrhizal mycelium was one to two orders of magnitude lower. The significance of this transfer as a 'short cut' in nutrient cycling is discussed.     

Ceratophyllum demersum hampers phytoplankton development in some small Norwegian lakes over a wide range of phosphorus concentrations and geographical latitude

1. Data on submerged and floating-leafed macrophytes, phytoplankton, nutrients (N, P) and calcium were collected from twenty-four small lakes ( 1 km²) over a wide range of latitudes in Norway. The majority of the investigated lakes were mesotrophic or eutrophic, and most of the lakes were markedly affected by diffuse and point-source runoff from agriculture. According to their macrophyte species composition, the majority of the lakes can be classified as Potamogeton lakes or Chara lakes, or a combination of these.
2. This study is consistent with the 'two alternative stable states' hypothesis. We observed clearwater lakes with dense macrophyte cover over a wider range of total P concentration than has been reported previously: from 30 to more than 700 mg P m^–3. The clearwater state was only observed in lakes with mean depths of less than 1.9 m.
3. Most clear lakes with high cover of submerged vegetation showed indications of N limitation.
4. In this study nearly all the macrophyte-dominated lakes with P concentrations above 30 mg m^–3 had dense stands of Ceratophyllum demersum (L.). This indicates that Ceratophyllum may also play an important role in stabilizing and maintaining a clearwater state at high P concentrations.     

Demographic and genetic effects on reproduction as related to population size in a rare, perennial herb, Senecio integrifolius (Asteraceae)

Seed-set of the rare and threatened plant Senecio integrifolius increased significantly with population size. Experimental studies as well as field observations showed this to be due to density-dependent seed-set (Allee effect). Hand-pollination revealed lower seed-set, and a lower germination rate of inbred progeny than of outbred progeny, with great differences among populations. Contrary to general predictions in models of minimum viable population sizes, the present study indicates little negative effects of inbreeding in small populations. A genetic load model was invoked to explain the results, hypothesizing that purging of deleterious alleles in small populations has reduced inbreeding depression. However, no clear correlation between population size and genetic load was found. The results in this paper suggest that demographic and environmental factors are of greater immediate importance than population genetics in determining extinction probabilities of small plant populations.     

Seasonal Effects on Photosynthetic Electron Transport and Fluorescence Properties in Isolated Chloroplasts of Pinus silvestris

Chloroplasts were isolated from primary needles of 1-year-old seedlings and from secondary needles of a 20-year-old pine tree in a natural stand. In autumn the electron transport capacities of PSII, PSI and PS (II + I) decreased and the electron transport between PSII and PSI became inhibited in October in the 20-year-old tree. This inhibition lasted until May the following year. The partial reactions of PSI and PSII still showed low but fairly constant rates during the whole winter seedlings. Seasonal changes in the electron transport properties of 1-year-old showed the same general trends as observed in the 20-year-old tree, but the changes were less pronounced. However, in snow-covered seedlings the PSI-mediated electron transport and the electron transport from H₂O to NADP increased during the late winter when the seedlings were still covered by snow. The total chlorophyll content of the needles decreased in autumn and winter. Low temperature fluorescence ratios of F692/F680 and F726/F680 indicated more severe destruction of the chlorophyll a antennae closely associated with the two photosystems than of the light harvesting chlorophyll a/b complex. In this case, too, the changes were more pronounced in the 20-year-old tree than in the 1-year-old seedlings. The chlorophyll/P700 ratios indicated a more marked reduction in the reaction centre molecules during autumn than in the antennae chlorophyll molecules. The changes in electron transport and low temperature fluorescence properties which occurred during autumn and winter were mainly reversed during spring.     

Galpha protein selectivity determinant specified by a viral chemokine receptor-conserved region in the C tail of the human herpesvirus 8 g protein-coupled receptor

The viral G-protein coupled receptor (vGPCR) specified by human herpesvirus 8 (HHV-8) open reading frame 74 (ORF74) is a ligand-independent chemokine receptor that has structural and functional homologues among other characterized gammaherpesviruses and related receptors in the betaherpesviruses. Sequence comparisons of the gammaherpesvirus vGPCRs revealed a highly conserved region in the C tail, just distal to the seventh transmembrane domain. Mutagenesis of the corresponding codons of HHV-8 ORF74 was carried out to provide C-tail-altered proteins for functional analyses. By measuring receptor-activated vascular endothelial growth factor promoter induction and NF-kappaB, mitogen-activated protein kinase, and Ca(2+) signaling, we found that while some altered receptors showed general signaling deficiencies, others had distinguishable activation profiles, suggestive of selective Galpha protein coupling. This was supported by the finding that vGPCR and representative functionally altered variants, vGPCR.8 (R322W) and vGPCR.15 (M325S), were affected differently by inhibitors of Galpha(i) (pertussis toxin), protein kinase C (GF109203X), and phosphatidylinositol 3-kinase (wortmannin). Consistent with the signaling data, [(35)S]GTPgammaS incorporation assays revealed preferential coupling of vGPCR.15 to Galpha(q) and an inability of vGPCR.8 to couple functionally to Galpha(q). However, both variants, wild-type vGPCR, and a C-tail deletion version of the receptor were equally able to associate physically with Galpha(q). Combined, our data demonstrate that HHV-8 vGPCR contains discrete sites of Galpha interaction and that receptor residues in the proximal region of the cytoplasmic tail are determinants of Galpha protein coupling specificity.