The role of P-type IIA and P-type IIB Ca2+-ATPases in plant development and growth.

As sessile organisms, plants have evolved mechanisms to adapt to variable and rapidly fluctuating environmental conditions. Calcium (Ca2+) in plant cells is a versatile intracellular second messenger that is essential for stimulating short- and long-term responses to environmental stresses through changes in its concentration in the cytosol ([Ca2+]cyt). Increases in [Ca2+]cyt direct the strength and length of these stimuli. In order to terminate them, the cells must then remove the cytosolic Ca2+ against a concentration gradient, either taking it away from the cell or storing it in organelles such as the endoplasmic reticulum (ER) and/or vacuoles. Here, we review current knowledge about the biological roles of plant P-type Ca2+-ATPases as potential actors in the regulation of this cytosolic Ca2+ efflux, with a focus the IIA ER-type Ca2+-ATPases (ECAs) and the IIB autoinhibited Ca2+-ATPases (ACAs). While ECAs are analogous proteins to animal sarcoplasmic-endoplasmic reticulum Ca2+-ATPases (SERCAs), ACAs are equivalent to animal plasma membrane-type ATPases (PMCAs). We examine their expression patterns in cells exhibiting polar growth and consider their appearance during the evolution of the plant lineage. Full details of the functions and coordination of ECAs and ACAs during plant growth and development have not yet been elucidated. Our current understanding of the regulation of fluctuations in Ca2+ gradients in the cytoplasm and organelles during growth is in its infancy, but recent technological advances in Ca2+ imaging are expected to shed light on this subject.

Calcium dynamics in tomato pollen tubes using the Yellow Cameleon 3.6 sensor.

KEY MESSAGE: In vitro tomato pollen tubes show a cytoplasmic calcium gradient that oscillates with the same period as growth. Pollen tube growth requires coordination between the tip-focused cytoplasmic calcium concentration ([Ca2+]cyt) gradient and the actin cytoskeleton. This [Ca2+]cyt gradient is necessary for exocytosis of small vesicles, which contributes to the delivery of new membrane and cell wall at the pollen tube tip. The mechanisms that generate and maintain this [Ca2+]cyt gradient are not completely understood. Here, we studied calcium dynamics in tomato (Solanum lycopersicum) pollen tubes using transgenic tomato plants expressing the Yellow Cameleon 3.6 gene under the pollen-specific promoter LAT52. We use tomato as an experimental model because tomato is a Solanaceous plant that is easy to transform, and has an excellent genomic database and genetic stock center, and unlike Arabidopsis, tomato pollen is a good system to do biochemistry. We found that tomato pollen tubes showed an oscillating tip-focused [Ca2+]cyt gradient with the same period as growth. Then, we used a pharmacological approach to disturb the intracellular Ca2+ homeostasis, evaluating how the [Ca2+]cyt gradient, pollen germination and in vitro pollen tube growth were affected. We found that cyclopiazonic acid (CPA), a drug that inhibits plant PIIA-type Ca2+-ATPases, increased [Ca2+]cyt in the subapical zone, leading to the disappearance of the Ca2+ oscillations and inhibition of pollen tube growth. In contrast, 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of Ca2+ released from the endoplasmic reticulum to the cytoplasm in animals cells, completely reduced [Ca2+]cyt at the tip of the tube, blocked the gradient and arrested pollen tube growth. Although both drugs have antagonistic effects on [Ca2+]cyt, both inhibited pollen tube growth triggering the disappearance of the [Ca2+]cyt gradient. When CPA and 2-APB were combined, their individual inhibitory effects on pollen tube growth were partially compensated. Finally, we found that GsMTx-4, a peptide from spider venom that blocks stretch-activated Ca2+ channels, inhibited tomato pollen germination and had a heterogeneous effect on pollen tube growth, suggesting that these channels are also involved in the maintenance of the [Ca2+]cyt gradient. All these results indicate that tomato pollen tube is an excellent model to study calcium dynamics.

Cross-Species Network Analysis Uncovers Conserved Nitrogen-Regulated Network Modules in Rice.

In this study, we used a cross-species network approach to uncover nitrogen (N)-regulated network modules conserved across a model and a crop species. By translating gene network knowledge from the data-rich model Arabidopsis (Arabidopsis thaliana) to a crop, rice (Oryza sativa), we identified evolutionarily conserved N-regulatory modules as targets for translational studies to improve N use efficiency in transgenic plants. To uncover such conserved N-regulatory network modules, we first generated an N-regulatory network based solely on rice transcriptome and gene interaction data. Next, we enhanced the network knowledge in the rice N-regulatory network using transcriptome and gene interaction data from Arabidopsis and new data from Arabidopsis and rice plants exposed to the same N treatment conditions. This cross-species network analysis uncovered a set of N-regulated transcription factors (TFs) predicted to target the same genes and network modules in both species. Supernode analysis of the TFs and their targets in these conserved network modules uncovered genes directly related to N use (e.g. N assimilation) and to other shared biological processes indirectly related to N. This cross-species network approach was validated with members of two TF families in the supernode network, BASIC-LEUCINE ZIPPER TRANSCRIPTION FACTOR1-TGA and HYPERSENSITIVITY TO LOW PI-ELICITED PRIMARY ROOT SHORTENING1 (HRS1)/HRS1 Homolog family, which have recently been experimentally validated to mediate the N response in Arabidopsis.

The histone methyltransferase SDG8 mediates the epigenetic modification of light and carbon responsive genes in plants.

BACKGROUND: Histone methylation modifies the epigenetic state of target genes to regulate gene expression in the context of developmental and environmental changes. Previously, we used a positive genetic screen to identify an Arabidopsis mutant, cli186, which was impaired in carbon and light signaling. Here, we report a deletion of the Arabidopsis histone methyltransferase SDG8 in this mutant (renamed sdg8-5), which provides a unique opportunity to study the global function of a specific histone methyltransferase within a multicellular organism. RESULTS: To assess the specific role of SDG8, we examine how the global histone methylation patterns and transcriptome were altered in the sdg8-5 deletion mutant compared to wild type, within the context of transient light and carbon treatments. Our results reveal that the sdg8 deletion is associated with a significant reduction of H3K36me3, preferentially towards the 3' end of the gene body, accompanied by a reduction in gene expression. We uncover 728 direct targets of SDG8 that have altered methylation in the sdg8-5 mutant and are also bound by SDG8. As a group, this set of SDG8 targets is enriched in specific biological processes including defense, photosynthesis, nutrient metabolism and energy metabolism. Importantly, 64% of these SDG8 targets are responsive to light and/or carbon signals. CONCLUSIONS: The histone methyltransferase SDG8 functions to regulate the H3K36 methylation of histones associated with gene bodies in Arabidopsis. The H3K36me3 mark in turn is associated with high-level expression of a specific set of light and/or carbon responsive genes involved in photosynthesis, metabolism and energy production.

Modeling the global effect of the basic-leucine zipper transcription factor 1 (bZIP1) on nitrogen and light regulation in Arabidopsis.

BACKGROUND: Nitrogen and light are two major regulators of plant metabolism and development. While genes involved in the control of each of these signals have begun to be identified, regulators that integrate gene responses to nitrogen and light signals have yet to be determined. Here, we evaluate the role of bZIP1, a transcription factor involved in light and nitrogen sensing, by exposing wild-type (WT) and bZIP1 T-DNA null mutant plants to a combinatorial space of nitrogen (N) and light (L) treatment conditions and performing transcriptome analysis. We use ANOVA analysis combined with clustering and Boolean modeling, to evaluate the role of bZIP1 in mediating L and N signaling genome-wide. RESULTS: This transcriptome analysis demonstrates that a mutation in the bZIP1 gene can alter the L and/or N-regulation of several gene clusters. More surprisingly, the bZIP1 mutation can also trigger N and/or L regulation of genes that are not normally controlled by these signals in WT plants. This analysis also reveals that bZIP1 can, to a large extent, invert gene regulation (e.g., several genes induced by N in WT plants are repressed by N in the bZIP1 mutant). CONCLUSION: These findings demonstrate that the bZIP1 mutation triggers a genome-wide de-regulation in response to L and/or N signals that range from i) a reduction of the L signal effect, to ii) unlocking gene regulation in response to L and N combinations. This systems biology approach demonstrates that bZIP1 tunes L and N signaling relationships genome-wide, and can suppress regulatory mechanisms hypothesized to be needed at different developmental stages and/or environmental conditions.

Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1.

Understanding how nutrients affect gene expression will help us to understand the mechanisms controlling plant growth and development as a function of nutrient availability. Nitrate has been shown to serve as a signal for the control of gene expression in Arabidopsis. There is also evidence, on a gene-by-gene basis, that downstream products of nitrogen (N) assimilation such as glutamate (Glu) or glutamine (Gln) might serve as signals of organic N status that in turn regulate gene expression. To identify genome-wide responses to such organic N signals, Arabidopsis seedlings were transiently treated with ammonium nitrate in the presence or absence of MSX, an inhibitor of glutamine synthetase, resulting in a block of Glu/Gln synthesis. Genes that responded to organic N were identified as those whose response to ammonium nitrate treatment was blocked in the presence of MSX. We showed that some genes previously identified to be regulated by nitrate are under the control of an organic N-metabolite. Using an integrated network model of molecular interactions, we uncovered a subnetwork regulated by organic N that included CCA1 and target genes involved in N-assimilation. We validated some of the predicted interactions and showed that regulation of the master clock control gene CCA1 by Glu or a Glu-derived metabolite in turn regulates the expression of key N-assimilatory genes. Phase response curve analysis shows that distinct N-metabolites can advance or delay the CCA1 phase. Regulation of CCA1 by organic N signals may represent a novel input mechanism for N-nutrients to affect plant circadian clock function.

Functional analysis of the metallothionein gene cgMT1 isolated from the actinorhizal tree Casuarina glauca.

cgMT1 is a metallothionein (MT)-like gene that was isolated from a cDNA library of young nitrogen-fixing nodules resulting from the symbiotic interaction between Frankia spp. and the actinorhizal tree Casuarina glauca. cgMT1 is highly transcribed in the lateral roots and nitrogen-fixing cells of actinorhizal nodules; it encodes a class I type 1 MT. To obtain insight into the function of cgMT1, we studied factors regulating the expression of the MT promoter region (PcgMT1) using a beta-glucuronidase (gus) fusion approach in transgenic plants of Arabidopsis thaliana. We found that copper, zinc, and cadmium ions had no significant effect on the regulation of PcgMT1-gus expression whereas wounding and H2O2 treatments led to an increase in reporter gene activity in transgenic leaves. Strong PcgMT1-gus expression also was observed when transgenic plants were inoculated with a virulent strain of the bacterial pathogen Xanthomonas campestris pv. campestris. Transgenic Arabidopsis plants expressing cgMT1 under the control of the constitutive 35S promoter were characterized by reduced accumulation of H2O2 when leaves were wounded and by increased susceptibility to the bacterial pathogen X. campestris. These results suggest that cgMT1 could play a role during the oxidative response linked to biotic and abiotic stresses.

Comparison of four constitutive promoters for the expression of transgenes in the tropical nitrogen-fixing tree Allocasuarina verticillata.

Allocasuarina verticillata is an actinorhizal tree that lives in symbiotic association with a nitrogen fixing actinomycete called Frankia. In the search for promoters that drive strong constitutive expression in this tropical tree, we studied the organ specificity of four different constitutive promoters (CaMV 35S, e35S, e35S-4ocs and UBQ1 from Arabidopsis thaliana) in stably transformed A. verticillata plants. The ss-glucuronidase (gus) gene was used as a reporter and expression studies were carried out by histochemical analyses on shoots, roots and actinorhizal nodules. While the 35S promoter was poorly expressed in the shoot apex and lateral roots, both the e35S and e35S-4ocs were found to drive high constitutive expression in the transgenic non-nodulated plants. In contrast, the UBQ1 promoter was very poorly expressed and appeared unsuitable for A. verticillata. We also showed that none of the promoters studied were active in the nodule infected cells, whatever the developmental stage studied.

Interactions between Trichoderma pseudokoningii strains and the arbuscular mycorrhizal fungi Glomus mosseae and Gigaspora rosea.

The interaction between Trichoderma pseudokoningii (Rifai) 511, 2212, 741A, 741B and 453 and the arbuscular mycorrhizal fungi Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe BEG12 and Gigaspora rosea Nicolson & Schenck BEG9 were studied in vitro and in greenhouse experiments. All T. pseudokoningii strains inhibited the germination of G. mosseae and Gi. rosea except the strain 453, which did not affect the germination of Gi. rosea. Soluble exudates and volatile substances produced by all T. pseudokoningii strains inhibited the spore germination of G. mosseae. The germination of Gi. rosea spores was inhibited by the soluble exudates produced by T. pseudokoningii 2212 and 511, whereas T. pseudokoningii 714A and 714B inhibited the germination of Gi. rosea spores by the production of volatile substances. The strains of T. pseudokoningii did not affect dry matter and percentage of root length colonization of soybean inoculated with G. mosseae, except T. pseudokoningii 2212, which inhibited both parameters. However, all T. pseudokoningii strains decreased the shoot dry matter and the percentage of AM root length colonization of soybean inoculated with Gi. rosea. The saprotrophic fungi tested seem to affect AM colonization of root by effects on the presymbiotic phase of the AM fungi. No influence of AM fungi on the number of CFUs of T. pseudokoningii was found. The effect of saprotrophic fungi on AM fungal development and function varied with the strain of the saprotrophic species tested.