Ivo Rieu

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.

KNOX Action in Arabidopsis Is Mediated by Coordinate Regulation of Cytokinin and Gibberellin Activities

The shoot apical meristem (SAM) is a pluripotent group of cells that gives rise to the aerial parts of higher plants. Class-I KNOTTED1-like homeobox (KNOX) transcription factors promote meristem function partly through repression of biosynthesis of the growth regulator gibberellin (GA). However, regulation of GA activity cannot fully account for KNOX action. Here, we show that KNOX function is also mediated by cytokinin (CK), a growth regulator that promotes cell division and meristem function. We demonstrate that KNOX activity is sufficient to rapidly activate both CK biosynthetic gene expression and a SAM-localized CK-response regulator. We also show that CK signaling is necessary for SAM function in a weak hypomorphic allele of the KNOX gene SHOOTMERISTEMLESS (STM). Additionally, we provide evidence that a combination of constitutive GA signaling and reduced CK levels is detrimental to SAM function. Our results indicate that CK activity is both necessary and sufficient for stimulating GA catabolic gene expression, thus reinforcing the low-GA regime established by KNOX proteins in the SAM. We propose that KNOX proteins may act as general orchestrators of growth-regulator homeostasis at the shoot apex of Arabidopsis by simultaneously activating CK and repressing GA biosynthesis, thus promoting meristem activity.

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

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.

Perspectives on deciphering mechanisms underlying plant heat stress response and thermotolerance

Global warming is a major threat for agriculture and food safety and in many cases the negative effects are already apparent. The current challenge of basic and applied plant science is to decipher the molecular mechanisms of heat stress response (HSR) and thermotolerance in detail and use this information to identify genotypes that will withstand unfavorable environmental conditions. Nowadays X-omics approaches complement the findings of previous targeted studies and highlight the complexity of HSR mechanisms giving information for so far unrecognized genes, proteins and metabolites as potential key players of thermotolerance. Even more, roles of epigenetic mechanisms and the involvement of small RNAs in thermotolerance are currently emerging and thus open new directions of yet unexplored areas of plant HSR. In parallel it is emerging that although the whole plant is vulnerable to heat, specific organs are particularly sensitive to elevated temperatures. This has redirected research from the vegetative to generative tissues. The sexual reproduction phase is considered as the most sensitive to heat and specifically pollen exhibits the highest sensitivity and frequently an elevation of the temperature just a few degrees above the optimum during pollen development can have detrimental effects for crop production. Compared to our knowledge on HSR of vegetative tissues, the information on pollen is still scarce. Nowadays, several techniques for high-throughput X-omics approaches provide major tools to explore the principles of pollen HSR and thermotolerance mechanisms in specific genotypes. The collection of such information will provide an excellent support for improvement of breeding programs to facilitate the development of tolerant cultivars. The review aims at describing the current knowledge of thermotolerance mechanisms and the technical advances which will foster new insights into this process.

Gibberellin metabolism and signaling.

Gibberellins (GAs) are a family of plant hormones controlling many aspects of plant growth and development including stem elongation, germination, and the transition from vegetative growth to flowering. Cloning of the genes encoding GA biosynthetic and inactivating enzymes has led to numerous insights into the developmental regulation of GA hormone accumulation that is subject to both positive and negative feedback regulation. Genetic and biochemical analysis of GA-signaling genes has revealed that posttranslational regulation of DELLA protein accumulation is a key control point in GA response. The highly conserved DELLA proteins are a family of negative regulators of GA signaling that appear subject to GA-stimulated degradation through the ubiquitin-26S proteasome pathway. This review discusses the regulation of GA hormone accumulation and signaling in the context of its role in plant growth and development.

Ethylene regulates the timing of anther dehiscence in tobacco

We investigated the involvement of ethylene signaling in the development of the reproductive structures in tobacco (Nicotiana tabacum L.) by studying flowers that were insensitive to ethylene. Ethylene-insensitivity was generated either by expression of the mutant etr1-1 ethylene-receptor allele from Arabidopsis thaliana or by treatment with the ethylene-perception inhibitor 1-methylcyclopropene (MCP). Development of ovaries and ovules was unaffected by ethylene-insensitivity. Anther development was also unaffected, but the final event of dehiscence was delayed and was no longer synchronous with flower opening. We showed that in these anthers degeneration of the stomium cells and dehydration were delayed. In addition, we found that MCP-treatment of detached flowers and isolated, almost mature anthers delayed dehiscence whereas ethylene-treatment accelerated dehiscence. This indicated that ethylene has a direct effect on a process that takes place in the anthers just before dehiscence. Because a similar function has been described for jasmonic acid in Arabidopsis, we suggest that ethylene acts similarly to or perhaps even in concurrence with jasmonic acid as a signaling molecule controlling the processes that lead to anther dehiscence in tobacco.

How plants handle multiple stresses: hormonal interactions underlying responses to abiotic stress and insect herbivory

Adaptive plant responses to specific abiotic stresses or biotic agents are fine-tuned by a network of hormonal signaling cascades, including abscisic acid (ABA), ethylene, jasmonic acid (JA) and salicylic acid. Moreover, hormonal cross-talk modulates plant responses to abiotic stresses and defenses against insect herbivores when they occur simultaneously. How such interactions affect plant responses under multiple stresses, however, is less understood, even though this may frequently occur in natural environments. Here, we review our current knowledge on how hormonal signaling regulates abiotic stress responses and defenses against insects, and discuss the few recent studies that attempted to dissect hormonal interactions occurring under simultaneous abiotic stress and herbivory. Based on this we hypothesize that drought stress enhances insect resistance due to synergistic interactions between JA and ABA signaling. Responses to flooding or waterlogging involve ethylene signaling, which likely reduces plant resistance to chewing herbivores due to its negative cross-talk with JA. However, the outcome of interactions between biotic and abiotic stress signaling is often plant and/or insect species-dependent and cannot simply be predicted based on general knowledge on the involvement of signaling pathways in single stress responses. More experimental data on non-model plant and insect species are needed to reveal general patterns and better understand the molecular mechanisms allowing plants to optimize their responses in complex environments.

Ensuring Reproduction at High Temperatures: The Heat Stress Response during Anther and Pollen Development

Sexual reproduction in flowering plants is very sensitive to environmental stresses, particularly to thermal insults which frequently occur when plants grow in field conditions in the warm season. Although abnormalities in both male and female reproductive organs due to high temperatures have been described in several crops, the failure to set fruits has mainly been attributed to the high sensitivity of developing anthers and pollen grains, particularly at certain developmental stages. A global view of the molecular mechanisms involved in the response to high temperatures in the male reproductive organs will be presented in this review. In addition, transcriptome and proteomic data, currently available, will be discussed in the light of physiological and metabolic changes occurring during anther and pollen development. A deep understanding of the molecular mechanisms involved in the stress response to high temperatures in flowers and, particularly, in the male reproductive organs will be a major step towards development of effective breeding strategies for high and stable production in crop plants.

Ethylene-induced Arabidopsis hypocotyl elongation is dependent on but not mediated by gibberellins

Ethylene, or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), can stimulate hypocotyl elongation in the light. It is questioned whether gibberellins (GAs) play a role in this response. Tests with light of different wavelengths demonstrated that the ethylene response depends on blue light and functional cryptochrome signalling. Levels of bio-active GA(4) were reduced in seedlings showing an ethylene response. Furthermore, ACC treatment of seedlings caused accumulation of the DELLA protein RGA, a repressor of growth. Concurrently, transcript levels of several GA biosynthesis genes were up-regulated and GA inactivation genes down-regulated by ACC. Hypocotyl elongation in response to ACC was strongly reduced in seedlings with a diminished GA signal, while being vigorously stimulated in a quadruple DELLA knock-out mutant with constitutive GA signalling. These data show that ethylene-driven hypocotyl elongation is mainly blue light-dependent and that this ethylene response, although GA dependent, hence needing a basal GA level, is not mediated by GA, but rather acts via a separate pathway.