Anne-Marie Faber

Microanalysis of Plant Mitochondrial Protein-synthesis Products - Detection of Variant Polypeptides Associated With Cytoplasmic Male-sterility

SummaryA mitochondrial fraction obtained from 0.5 g of leaves was purified on a 0.75 ml Percoll gradient and used for an in vitro mitochondrial protein synthesis assay in the presence of [35S] methionine. A set of 15 to 20 labeled polypeptides were revealed by autoradiography after sodium dodecylsulfate polyacrylamide gel electrophoresis. This could be applied at an early growth stage by using a few leaves from individual seedlings. It revealed the presence of variant mitochondrially translated polypeptides in green leaves of cytoplasmic male sterile lines from various cultivated plants of large economic importance: maize, wheat, sugar beet, tobacco and faba bean. This non-destructive microanalysis is thus of general use and opens new possibilities for rapid and large mass screening of mitochondrial parameters such as male sterility.

Gel-based and gel-free proteomic analysis of Nicotiana tabacum trichomes identifies proteins involved in secondary metabolism and in the (a)biotic stress response

Nicotiana tabacum leaves are covered by trichomes involved in the secretion of large amounts of secondary metabolites, some of which play a major role in plant defense. However, little is known about the metabolic pathways that operate in these structures. We undertook a proteomic analysis of N. tabacum trichomes in order to identify their protein complement. Efficient trichome isolation was obtained by abrading frozen leaves. After homogenization, soluble proteins and a microsomal fraction were prepared by centrifugation. Gel‐based and gel‐free proteomic analyses were then performed. 2‐DE analysis of soluble proteins led to the identification of 1373 protein spots, which were digested and analyzed by MS/MS, leading to 680 unique identifications. Both soluble proteins and microsomal fraction were analyzed by LC MALDI‐MS/MS after trypsin digestion, leading to 858 identifications, many of which had not been identified after 2‐DE, indicating that the two methods complement each other. Many enzymes putatively involved in secondary metabolism were identified, including enzymes involved in the synthesis of terpenoid precursors and in acyl sugar production. Several transporters were also identified, some of which might be involved in secondary metabolite transport. Various (a)biotic stress response proteins were also detected, supporting the role of trichomes in plant defense.

Proteomic analysis of chicory root identifies proteins typically involved in cold acclimation.

Chicory (Cichorium intybus) roots contain high amounts of inulin, a fructose polymer used as a storage carbohydrate by the plant and as a human dietary and prebiotic compound. We performed 2‐D electrophoretic analysis of proteins from root material before the first freezing period. The proteins were digested with trypsin and the peptides analyzed by MS (MALDI‐TOF/TOF). From the 881 protein spots analyzed, 714 proteins corresponded to a database accession, 619 of which were classified into functional categories. Besides expected proteins (e.g. related to metabolism, energy, protein synthesis, or cell structure), other well‐represented categories were proteins related to folding and stability (49 spots), proteolysis (49 spots), and the stress response (67 spots). The importance of abiotic stress response was confirmed by the observation that 7 of the 21 most intense protein spots are known to be involved in cold acclimation. These results suggest a major effect of the low temperature period that preceded root harvesting.

The proteome complement of Nicotiana tabacum Bright-Yellow-2 culture cells.

The Nicotiana tabacum Bright‐Yellow‐2 (BY2) cell line is one of most commonly used plant suspension cell lines and offers interesting properties, such as fast growth, amenability to genetic transformation, and synchronization of cell division. To build a proteome reference map of BY2 cell proteins, we isolated the soluble proteins from N. tabacum BY2 cells at the end of the exponential growth phase and analyzed them by 2‐DE and MALDI TOF‐TOF. Of the 1422 spots isolated, 795 were identified with a significant score, corresponding to 532 distinct proteins.

Identification of 3 Distinct Spectral Species of Yeast Mitochondrial Cytochrome-b Using a Combination of Respiratory Inhibitors

Abstract Appropriate combination of specific inhibitors of electron transport in the cytochrome bc 1 segment of the respiratory chain of Saccharomyces cerevisiae allows the rapid resolution of three spectral forms of mitochondrial cytochrome b . (1) Addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to aerobic yeast submitochondrial particles preincubated with cyanide and mucidin in the presence of NADH reveals cytochrome b -561.5. (2) Addition of funiculosin to aerobic yeast submitochondrial particles preincubated with cyanide, mucidin and n -heptylhydroxyquinoline N -oxide in the presence of NADH reveals cytochrome b -558 independently of cytochrome b -561.5 and cytochrome b -565. (3) Specific resolution of cytochrome b -565 can be obtained either by addition of mucidin to aerobic submitochondrial particles preincubated with cyanide, DCMU and NADH, or by addition of antimycin plus an oxygen pulse to NADH-reduced particles, preincubated with cyanide, in the presence of ascorbate plus TMPD, or by addition of antimycin A in the presence of oxidized TMPD to aerobically NADH-reduced particles.

Toward a better understanding of the mechanisms of symbiosis: a comprehensive proteome map of a nascent insect symbiont

Symbiotic bacteria are common in insects and can affect various aspects of their hosts’ biology. Although the effects of insect symbionts have been clarified for various insect symbiosis models, due to the difficulty of cultivating them in vitro, there is still limited knowledge available on the molecular features that drive symbiosis. Serratia symbiotica is one of the most common symbionts found in aphids. The recent findings of free-living strains that are considered as nascent partners of aphids provide the opportunity to examine the molecular mechanisms that a symbiont can deploy at the early stages of the symbiosis (i.e., symbiotic factors). In this work, a proteomic approach was used to establish a comprehensive proteome map of the free-living S. symbiotica strain CWBI-2.3T. Most of the 720 proteins identified are related to housekeeping or primary metabolism. Of these, 76 were identified as candidate proteins possibly promoting host colonization. Our results provide strong evidence that S. symbiotica CWBI-2.3T is well-armed for invading insect host tissues, and suggest that certain molecular features usually harbored by pathogenic bacteria are no longer present. This comprehensive proteome map provides a series of candidate genes for further studies to understand the molecular cross-talk between insects and symbiotic bacteria.