Effect of jasmonic acid on developmental morphology during in vitro tuberization of Dioscorea alata (L.)

A developmental morphology study was performed during different stages of in vitro yam microtuber formation on both hormone-free medium (HF) and medium supplemented with 10 µM of jasmonic acid (JA). The axillary protuberance, considered as the first morphological evidence for microtuber formation, became visible after one-week of culture in JA-medium and after three weeks of culture in HF-medium. In addition, the formation of the axillary protuberance in JA-cultures preceded the stem elongation, whereas in HF-medium stem elongation was visible before the formation of the axillary protuberance. Tuberization improvement in medium supplemented with JA is likely to be the result of morphogenetic modifications occurring during the early stages of microtuber formation. At the molecular level, 2D-PAGE analysis of HF- and JA-cultures allowed the identification of four differentially expressed polypeptides, including two putative pathogenesis-related proteins and one putative glutathione S-transferase. For JA-cultures, three additional differentially expressed polypeptides were identified.     

High resolution synteny maps allowing direct comparisons between the coffee and tomato genomes

Tomato (Solanum lycopersicum) and coffee (Coffea canephora) belong to the sister families Solanaceae and Rubiaceae, respectively. We report herein the mapping of a common set of 257 Conserved Ortholog Set II genes in the genomes of both species. The mapped markers are well distributed across both genomes allowing the first syntenic comparison between species from these two families. The majority (75%) of the synteny blocks are short (<4 cM); however, some extend up to 50 cM. In an effort to further characterize the synteny between these two genomes, we took advantage of the available sequence for the tomato genome to show that tomato chromosome 7 is syntenic to half of the two coffee linkage groups E and F with the putative break point in tomato localized to the boundary of the heterochromatin and euchromatin on the long arm. In addition to the new insight on genome conservation and evolution between the plant families Solanaceae and Rubiaceae, the comparative maps presented herein provide a translational tool by which coffee researchers may take benefit of DNA sequence and genetic information from tomato and vice versa. It is thus expected that these comparative genome information will help to facilitate and expedite genetic and genomic research in coffee.     

Fatty acid hydroperoxides biotransformation by potato tuber cell-free extracts

Cleavage of 13-HPOD, 13-HPOT, 9-HPOD and 9-HPOT by potato tuber cell-free extracts was investigated. 13-HPOD and 13-HPOT enzymes were degraded almost completely while 9-HPOD and 9-HPOT were partially transformed. GC-MS analysis of the volatile compounds formed during the reactions revealed that (Z)-3 hexenal, (E)-2-hexenal, pentenols and dimers of pentene were obtained from 13-HPOT while from 13-HPOD hexanal and pentan-1-ol were formed. No volatile was found when 9-HPO isomers were used as substrate, but colneleic acid was produced. When Triton X-100 was omitted in the extraction buffer, only pentenols and dimers of pentene were identified from 13-HPOT and pentan-1-ol from 13-HPOD. Our results reveal that potato tubers that contain Lox, which forms mainly 9-HPO, are able to metabolise the four HPO isomers. Moreover, 13-HPO cleaving activities are due to two distinct enzymatic systems based on, respectively, homolytic and heterolytic mechanisms. The fact that oxygenation of reaction medium dramatically decreases the amount of product resulting from homolytic cleavage strengthens the hypothesis of an anaerobic reaction due to Lox.     

Enzymatic detoxification of eutypine, a toxin from Eutypa lata, by Vitis vinifera cells: partial purification of an NADPH-dependent aldehyde reductase

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata (Pers.: Fr.) Tul., the causal agent of dying arm disease of Vitis vinifera L. (grapevine). Previously, we have shown that eutypine is involved in the development of disease symptoms. In the present study, the effects of V. vinifera cell-suspension cultures on the biological activity of the toxin were investigated. Eutypine was converted by grapevine tissues into a single compound, identified by mass spectrometry and nuclear magnetic resonance as 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl alcohol, designated eutypinol. This compound was found to be non-toxic for grapevine tissues. Unlike eutypine, eutypinol failed to affect the oxidation rate or membrane potential of isolated mitochondria. In grapevine cells, reduction of eutypine into the corresponding alcohol is an NADPH-dependent enzymatic reaction. An enzyme which reduced eutypine was partially purified, over 1000-fold, using a five-step purification procedure. By gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was found to have a molecular mass of 54–56 kDa. The enzyme exhibited an apparent K _m for eutypine of 44 μM, and was active between pH 6.8 and 7.5 with a maximum at pH 7.0. The eutypine reductase activity was improved by Mn²⁺ and Mg²⁺ and inhibited by disulfiram and p-hydroxymercuribenzoate. The possible role of the eutypine-detoxification mechanism in the defense reactions of V. vinifera cells is discussed. Received: 20 April 1998 / Accepted: 22 September 1998     

A novel NADPH-dependent aldehyde reductase gene from Vigna radiata confers resistance to the grapevine fungal toxin eutypine

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl aldehyde, is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevines. It has previously been demonstrated that tolerance of some cultivars to this disease was correlated with their capacity to convert eutypine to the corresponding alcohol, eutypinol, which lacks phytotoxicity. We have thus purified to homogeneity a protein from Vigna radiata that exhibited eutypine-reducing activity and have isolated the corresponding cDNA. This encodes an NADPH-dependent reductase of 36 kDa that we have named Vigna radiata eutypine-reducing enzyme (VR-ERE), based on the capacity of a recombinant form of the protein to reduce eutypine into eutypinol. The strongest homologies (86.8%) of VR-ERE at the amino acid level were found with CPRD14, a drought-inducible gene of unknown function, isolated from Vigna unguiculata and with an aromatic alcohol dehydrogenase (71.7%) from Eucalyptus gunnii . Biochemical characterization of VR-ERE revealed that a variety of compounds containing an aldehyde group can act as substrates. However, the highest affinity was observed with 3-substituted benzaldehydes. Expression of a VR-ERE transgene in Vitis vinifera cells cultured in vitro conferred resistance to the toxin. This discovery opens up new biotechnological approaches for the generation of grapevines resistant to eutypa dieback.     

TdERF1, an ethylene response factor associated with dehydration responses in durum wheat (Triticum turgidum L. subsp. durum)

Water deficit and increasing salinization reduce productivity of wheat, the leading crop for human diet. While the complete genome sequence of this crop has not been deciphered, a BAC library screening allowed the isolation of TdERF1, the first ethylene response factor gene from durum wheat. This gene is putatively involved in mediating salt stress tolerance and its characterization provides clues toward understanding the mechanisms underlying the adaptation/tolerance of durum wheat to suboptimal growth conditions. TdERF1 expression is differentially induced by high salt treatment in 2 durum wheat varieties, the salt-tolerant Grecale (GR) and the salt-sensitive Om Rabiaa (OR). To further extend these findings, we show here that the expression of this ERF is correlated with physiological parameters, such as the accumulation of osmo-regulators and membrane integrity, that discriminate between the 2 contrasted wheat genotypes. The data confirm that GR and OR are 2 contrasted wheat genotypes with regard to salt-stress and show that TdERF1 is also induced by water stress with an expression pattern clearly discriminating between the 2 genotypes. These findings suggest that TdERF1 might be involved in responses to salt and water stress providing a potential genetic marker discriminating between tolerant and sensitive wheat varieties.     

Towards the development of multifunctional molecular indicators combining soil biogeochemical and microbiological variables to predict the ecological integrity of silvicultural practices

The impact of mechanical site preparation (MSP) on soil biogeochemical structure in young larch plantations was investigated. Soil samples were collected in replicated plots comprising simple trenching, double trenching, mounding and inverting site preparation. Unlogged natural mixed forest areas were used as a reference. Analysis of soil nutrients, abundance of bacteria and gas exchanges unveiled no significant difference among the plots. However, inverting site preparation resulted in higher variations of gas exchanges when compared with trenching, mounding and unlogged natural forest. A combination of the biological and physicochemical variables was used to define a multifunctional classification of the soil samples into four distinct groups categorized as a function of their deviation from baseline ecological conditions. According to this classification model, simple trenching was the approach that represented the lowest ecological risk potential at the microsite level. No relationship was observed between MSP method and soil bacterial community structure as assessed by high‐throughput sequencing of bacterial 16S rRNA gene; however, indicator genotypes were identified for each multifunctional soil class. This is the first identification of multifunctional molecular indicators for baseline and disturbed ecological conditions in soil, demonstrating the potential of applied microbial ecology to guide silvicultural practices and ecological risk assessment.     

Subcellular localization of the sites of conversion of 1-aminocyclopropane-1-carboxylic acid into ethylene in plant cells

The subcellular localization of the sites of 1-aminocyclopropane-1-carboxylic acid (ACC) conversion into ethylene was studied by comparing the specific radioactivity of ethylene evolved from the whole cells with that of intra- and extracellular pools of labelled ACC. We demonstrate that some cells cultured in vitro (Vitis vinifera L. cv. Muscat) or leaf tissues (Hordeum vulgare L. and Triticum aestivum L.) have two sites of ethylene production: (i) an external site, converting apoplastic ACC, located at the plasma membrane, and very sensitive to high osmotica and, (ii) an intracellular site, converting internal ACC and remaining unaffected even under severe plasmolysis. In other cells cultured in vitro (Vitis vinifera L. cv. Gamay) and pea leaves (Pisum sativum L.), only the intracellular site operates and ethylene production is almost unaffected by plasmolysis. Protoplasts obtained from plasmolysis-sensitive Muscat cells lose 97% of their capacity for ethylene production compared with the parent cell, while those from plasmolysisinsensitive Gamay cells retain up to 50%. Protoplasts from both Gamay and Muscat cells cultured for 8 d in vitro, recover the full capacity of ethylene production of the initial whole cells, whether or not they are allowed to reform their cell wall. Therefore, we exclude a cooperation between the cell wall and the plasma membrane in ethylene production.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme We are grateful to Dr. Philip John (Reading, UK) for useful discus sions made possible by a North Atlantic Treaty Organization Colla borative Grant (No. 0383/88) and Dr. Yves Meyer (Perpignan, France) for his collaboration in culturing protoplasts.