No abstract

Eleven cDNA clones encoding the open-reading frame of calmodulin and calmodulin-like proteins were isolated from carrot seedlings treated with fungal mycelial walls. These calmodulin genes were mainly expressed in the stems and leaves of carrot, although the transcriptional level was almost negligible in the seeds and root tissues. Based on nucleotide homology, these genes were divided into two classes, class I and class II, and reverse transcription-polymerase chain reaction analyses revealed that the expression level of class II genes transiently increased when carrot seedlings were treated with the mycelial walls. In contrast, the level of class I genes apparently did not show any significant change even after treatment with the fungal components. These results suggest that the defense responses induced in the fungi-infected carrot accompany the increase in the transcriptional levels of a certain calmodulin gene(s).

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Production of carrot (Daucus carota L.) phytoalexin, 6-methoxymellein, is triggered by the contact of the cells with oligogalacturonide elicitor, and the increase in cytoplasmic Ca 2ϩ level and activation of Ca 2ϩ -cascade in the cells are essential events in the elicitation process. 1) We have also shown 2) that calmodulin (CAM) is a key modulator protein to evoke phytoalexin biosynthesis in oligouronide elicitortreated carrot. CAM had been considered to be a constitutively expressed Ca 2ϩ -binding protein, however, a series of recent studies clearly demonstrated 3,4) that, unlike in animal cells, plant CAM is encoded by several genes and the specific CAM gene is sometimes expressed by various physiological stresses. We have recently isolated four clones of CAM gene from carrot (GenBank accession number AY364011, AY364012, AY543014, and AY543015), and found that among them, the transcription level of cam-4 (AY364011) was markedly increased by the treatment of carrot cells with oligogalacturonide elicitor. 5) We assumed it possible that the transformation of plant cells with appropriate gene(s) encoding key protein(s) in signal transduction mechanisms of phytoalexin induction, such as CAM, would activate Ca 2ϩ -cascade and maintain the cells in the excitatory state; this might enhance the biosynthetic activity of secondary metabolites relating plant defense responses in the transformed plants. At present, only very limited information is available about the biochemical changes in the plant cells transformed with CAM gene, however, it has been reported 6,7) that over-expression of the gene in tobacco cells results in the unusual activation of CAM-dependent functional proteins followed by the generation of active oxygen species and NO. In animal cells it was shown 8) that the elevation of NO concentration appreciably enhances the synthesis of cyclic nucleotides. On the other hand, we showed 9,10) that the increase in cytoplasmic level of cyclic nucleotides stimulates Ca 2ϩ -cascade in cultured carrot cells by activating the nucleotide-sensitive cation channels. In the present study, we attempted to produce the transgenic plants in which carrot cam-4 gene was introduced and over-expressed by the infection of transformed Agrobacterium tumefaciens. Possible changes in the activity of key enzymes of phenylpropanoid metabolism, phenylalanine ammonia-lyase (PAL) and caffeic acid O-methyltransferase (COMT), in the transformed plants were examined together with the contents of phenolic compounds. In the preliminary experiments, sesame (Sesamum schinzianum ASCH.) showed the highest transformation and re-differentiation efficiencies among several plants tested, and we therefore focused on this plant as the target of the transformation. MATERALS AND METHODS Plant MaterialsSeeds of S. schinzianum were surfacesterilized in 70% (v/v) ethanol and 2% (v/v) sodium hypochlorite successively, and after several washings with autoclaved water they were placed on Murashige and Skoog's agar medium 11) for germination. The sterilized see...