A chemical complementation approach reveals genes and interactions of flavonoids with other pathways

In addition to the classical functions of flavonoids in the response to biotic/abiotic stress conditions, these phenolic compounds have been implicated in the modulation of various developmental processes. These findings suggest that flavonoids are more integral components of the plant signaling machinery than traditionally recognized. To understand how flux through the flavonoid pathway affects plant cellular processes, we used wild‐type and chalcone isomerase mutant (transparent testa 5, tt5) seedlings grown under anthocyanin inductive conditions, in the presence or absence of the flavonoid intermediate naringenin, the product of the chalcone isomerase enzyme. Because flavonoid biosynthetic genes are expressed under anthocyanin inductive conditions regardless of whether anthocyanins are formed or not, this system provides an excellent opportunity to specifically investigate the molecular changes associated with increased flux through the flavonoid pathway. By assessing genome‐wide mRNA accumulation changes in naringenin‐treated and untreated tt5 and wild‐type seedlings, we identified a flavonoid‐responsive gene set associated with cellular trafficking, stress responses and cellular signaling. Jasmonate biosynthetic genes were highly represented among the signaling pathways induced by increased flux through the flavonoid pathway. In contrast to studies showing a role for flavonoids in the control of auxin transport, no effect on auxin‐responsive genes was observed. Taken together, our data suggest that Arabidopsis can sense flavonoids as a signal for multiple fundamental cellular processes.     

Tomato tos1 mutation identifies a gene essential for osmotic tolerance and abscisic acid sensitivity

Osmotic stress severely limits plant growth and agricultural productivity. We have used mutagenesis to identify plant genes that are required for osmotic stress tolerance in tomato. As a result, we have isolated a novel mutant in tomato (tos1) caused by a single recessive nuclear mutation that is hypersensitive to general osmotic stress. Growth measurements demonstrated that the tos1 mutant is less sensitive to intracellular abscisic acid (ABA) and this decreased ABA sensitivity of tos1 is a basic cellular trait expressed by the mutant at all developmental stages analysed. It is not caused by a deficiency in the synthesis of ABA because the tos1 seedlings accumulated more ABA than the wild type (WT) after osmotic stress. In contrast, the tss2 tomato mutant, which is also hypersensitive to osmotic stress, is hypersensitive to exogenous ABA. Comparative analysis of tos1 and tss2 indicates that appropriate ABA perception and signalling is essential for osmotic tolerance.     

A cellulose synthase‐like protein is required for osmotic stress tolerance in Arabidopsis

Osmotic stress imposed by soil salinity and drought stress significantly affects plant growth and development, but osmotic stress sensing and tolerance mechanisms are not well understood. Forward genetic screens using a root‐bending assay have previously identified salt overly sensitive (sos) mutants of Arabidopsis that fall into five loci, SOS1 to SOS5. These loci are required for the regulation of ion homeostasis or cell expansion under salt stress, but do not play a major role in plant tolerance to the osmotic stress component of soil salinity or drought. Here we report an additional sos mutant, sos6‐1, which defines a locus essential for osmotic stress tolerance. sos6‐1 plants are hypersensitive to salt stress and osmotic stress imposed by mannitol or polyethylene glycol in culture media or by water deficit in the soil. SOS6 encodes a cellulose synthase‐like protein, AtCSLD5. Only modest differences in cell wall chemical composition could be detected, but we found that sos6‐1 mutant plants accumulate high levels of reactive oxygen species (ROS) under osmotic stress and are hypersensitive to the oxidative stress reagent methyl viologen. The results suggest that SOS6/AtCSLD5 is not required for normal plant growth and development but has a critical role in osmotic stress tolerance and this function likely involves its regulation of ROS under stress.     

A novel calmodulin-binding protein functions as a negative regulator of osmotic stress tolerance in Arabidopsis thaliana seedlings

A clone for a novel Arabidopsisthaliana calmodulin (CaM)-binding protein of 25 kDa (AtCaMBP25) has been isolated by using a radiolabelled CaM probe to screen a cDNA expression library derived from A. thaliana cell suspension cultures challenged with osmotic stress. The deduced amino acid sequence of AtCaMBP25 contains putative nuclear localization sequences and shares significant degree of similarity with hypothetical plant proteins only. Fusion of the AtCaMBP25 coding sequence to reporter genes targets the hybrid protein to the nucleus. Bacterially expressed AtCaMBP25 binds, in a calcium-dependent manner, to a canonical CaM but not to a less conserved isoform of the calcium sensor. AtCaMBP25 is encoded by a single-copy gene, whose expression is induced in Arabidopsis seedlings exposed to dehydration, low temperature or high salinity. Transgenic plants overexpressing AtCaMBP25 exhibits an increased sensitivity to both ionic (NaCl) and non-ionic (mannitol) osmotic stress during seed germination and seedling growth. By contrast, transgenic lines expressing antisense AtCaMBP25 are significantly more tolerant to mannitol and NaCl stresses than the wild type. Thus, the AtCaMBP25 gene functions as a negative effector of osmotic stress tolerance and likely participates in stress signal transduction pathways.     

Neurodegenerative mutants in Drosophila: a means to identify genes and mechanisms involved in human diseases?

There are 50 ways to leave your lover (Simon 1987) but many more to kill your brain cells. Several neurodegenerative diseases in humans, like Alzheimer’s disease, have been intensely studied but the underlying cellular and molecular mechanisms are still unknown for most of them. For those syndromes where associated gene products have been identified their biochemistry and physiological as well as pathogenic function is often still under debate. This is in part due to the inherent limitations of genetic analyses in humans and other mammals and therefore experimentally accessible invertebrate in vivo models, such as Caenorhabditis elegans and Drosophila melanogaster, have recently been introduced to investigate neurodegenerative syndromes. Several laboratories have used transgenic approaches in Drosophila to study the human genes associated with neurodegenerative diseases. This has added substantially to our understanding of the mechanisms leading to neurodegenerative diseases in humans. The isolation and characterization of Drosophila mutants, which display a variety of neurodegenerative phenotypes, also provide valuable insights into genes, pathways, and mechanisms causing neurodegeneration. So far only about two dozen such mutants have been described but already their characterization reveals an involvement of various cellular functions in neurodegeneration, ranging from preventing oxidative stress to RNA editing. Some of the isolated genes can already be associated with human neurodegenerative diseases and hopefully the isolation and characterization of more of these mutants, together with an analysis of homologous genes in vertebrate models, will provide insights into the genetic and molecular basis of human neurodegenerative diseases.     

Differential genetic interactions of yeast stress response MAPK pathways

Genetic interaction screens have been applied with great success in several organisms to study gene function and the genetic architecture of the cell. However, most studies have been performed under optimal growth conditions even though many functional interactions are known to occur under specific cellular conditions. In this study, we have performed a large‐scale genetic interaction analysis in Saccharomyces cerevisiae involving approximately 49 × 1,200 double mutants in the presence of five different stress conditions, including osmotic, oxidative and cell wall‐altering stresses. This resulted in the generation of a differential E‐MAP (or dE‐MAP) comprising over 250,000 measurements of conditional interactions. We found an extensive number of conditional genetic interactions that recapitulate known stress‐specific functional associations. Furthermore, we have also uncovered previously unrecognized roles involving the phosphatase regulator Bud14, the histone methylation complex COMPASS and membrane trafficking complexes in modulating the cell wall integrity pathway. Finally, the osmotic stress differential genetic interactions showed enrichment for genes coding for proteins with conditional changes in phosphorylation but not for genes with conditional changes in gene expression. This suggests that conditional genetic interactions are a powerful tool to dissect the functional importance of the different response mechanisms of the cell.     

<Emphasis Type="Italic">Physcomitrella patens</Emphasis> is highly tolerant against drought,salt and osmotic stress

In order to determine the degree of tolerance of the moss Physcomitrella patens to different abiotic stress conditions, we examined its tolerance against salt, osmotic and dehydration stress. Compared to other plants like Arabidopsis thaliana, P. patens exhibits a high degree of abiotic stress tolerance, making it a valuable source for the identification of genes effecting the stress adaptation. Plants that had been treated with NaCl tolerated concentrations up to 350 mM. Treatments with sorbitol revealed that plants are able to survive concentrations up to 500 mM. Furthermore, plants that had lost 92% water on a fresh-weight basis were able to recover successfully. For molecular analyses, a P. patens expressed sequence tag (EST) database was searched for cDNA sequences showing homology to stress-associated genes of seed plants and bacteria. 45 novel P. patens genes were identified and subjected to cDNA macroarray analyses to define their expression pattern in response to water deficit. Among the selected cDNAs, we were able to identify a set of genes that is specifically up-regulated upon dehydration. These genes encode proteins exerting their function in maintaining the integrity of the plant cell as well as proteins that are known to be members of signaling networks. The identified genes will serve as molecular markers and potential targets for future functional analyses.     

Comprehensive phenotypic analysis for identification of genes affecting growth under ethanol stress in Saccharomyces cerevisiae

We quantified the growth behavior of all available single gene deletion strains of budding yeast under ethanol stress. Genome-wide analyses enabled the extraction of the genes and determination of the functional categories required for growth under this condition. Statistical analyses revealed that the growth of 446 deletion strains under stress induced by 8% ethanol was defective. We classified these deleted genes into known functional categories, and found that many were important for growth under ethanol stress including several categories that have not been characterized, such as peroxisome. We also performed genome-wide screening under osmotic stress and identified 329 osmotic-sensitive strains. We excluded these strains from the 446 ethanol-sensitive strains to extract the genes whose deletion caused sensitivity to ethanol-specific (359 genes), osmotic-specific (242 genes), and both stresses (87 genes). We also extracted the functional categories that are specifically important for growth under ethanol stress. The genes and functional categories identified in the analysis might provide clues to improving ethanol stress tolerance among yeast cells.     

The uncoupling protein and the potassium channel are activated by hyperosmotic stress in mitochondria from durum wheat seedlings

The plant uncoupling mitochondrial protein (PUMP) and the plant mitochondrial potassium channel (PmitoK_ATP) are two recently discovered energy‐dissipating systems present in plant mitochondria, which may play a role as defence systems under environmental stress. To verify whether hyperosmotic stress affects the two dissipating systems in durum wheat (Triticum durum Desf.), their functioning was studied in early etiolated seedlings maintained under moderate and severe salt (NaCl) and osmotic (mannitol) stress. As measures of mitochondrial stress mitochondrial integrity, membrane potential maintenance and oxygen uptake coupled with ATP synthesis during succinate and proline oxidation were investigated. Both PUMP and PmitoK_ATP were activated under stress conditions. Activation was clearly evident even under moderate stress when proline oxidation was inhibited, although mitochondrial integrity and succinate oxidation were still unaffected. Under severe stress, which significantly affected all the tested indicators of mitochondrial integrity and functionality, PUMP and PmitoK_ATP activation was further enhanced. Interestingly, both systems were activated by reactive oxygen species and were able to control mitochondrial superoxide anion production. These results suggest that PUMP and PmitoK_ATP serve as early antioxidant defence systems in response to hyperosmotic stress and that they are involved in a prolonged response to stress.     

拟南芥基因芯片数据中非生物胁迫相关基因的挖掘

利用方差分析法从拟南芥芯片表达谱数据库挖掘非生物胁迫相关基因,并对这些基因进行GO注释分析,从而揭示非生物胁迫的生物学意义,发现非生物胁迫主要影响植物基因表达过程的转录调节和信号转导过程的磷酸化。同时对这些基因的上游启动子区域序列进行分析,挖掘非生物胁迫反应调控过程和适应过程的转录因子,发现植物非生物胁迫过程主要受bHLH—ZIP类和ZN—FINGER类C2H2型转录因子的调节。