Yuki Ichinose3-b, K arin T iem ann3, C laudia Schw enger-Erger3, K azuhiro T oyoda3 C, F rauke H e in 3, Thom as H an selle3, H olger C ornels3 and W olfgang B arz3 * a Institu t für B iochem ie und B iotechnologie d er Pflanzen, W estfälische W ilhelm s-U niversität M ünster, D-48143 M ünster, G erm an y b Science and Technology for E nergy C onversion, G ra d u ate School of N atural Science and Technology, O kayam a U niversity, O kayam a 700 -8 5 3 0 , Japan c L aborato ry of P lant Pathology & G en etic E ngineering, College of A griculture, O k ay am a U niversity, O kayam a 700-8530, Japan * A utor for co rresp o n d en ce and re p rin t requests. Fax: (+49) 2518328371. E-m ail: barz@ uni-m uenster.de Z. N aturforsch. 55c, 4 4 -5 4 (2000), received S ep tem b er 9/O ctober 28, 1999 P lant D efen ce G enes, G en e A ctivation, G ene Suppression, A scochyta rabiei, Cicer arietinum L.In response to the exogenous application of elicitors and attem p ted invasion by pathogens, plants exhibit a wide range of defense reactions. To un d erstan d the defense m echanism s at the level of gene activation and deactivation, differential screenings w ere p erform ed to iso late cD N A clones which are differentially expressed in p ath ogen-inoculated resistant chick pea plants and elicitor-treated cell cultures. A plenty of genes were isolated and arran g ed in 5 groups, nam ely d efen se-related pathw ays, signal transduction pathw ays, regulation of gene expression, catabolic pathw ays and prim ary m etabolism . M ost of these genes w ere activated although several genes w ere also fo u n d to be suppressed. We discuss the plausible functions of cD N A pro d u cts in plan t defense responses. The cD N A s provide a variety of tools to investigate m o lecular m echanism s o f defense responses and clearly reflect the massive g en o mic and m etabolic changes which occur during m anifestation of antim icrobial defense. 0939-5075/2000/0100-0044 $ 06.00 © 2000 Verlag der Zeitschrift für Naturforschung, Tübingen • www.znaturforsch.com • D Unauthenticated Download Date | 6/21/16 8:05 AM fence -a broad perspective. Physiol. Mol. Plant Pathol. 51, 3 4 7 -3 6 6 . Bourois M., M oore P., Ruel L., Grau Y., Heitzler P. and Simpson P. (1990), An early embryonic product of the gene shaggy encodes a serine/threonine protein ki nase related to the C D C 28/cdc2+ subfamily. E M B O J. 9, 2 8 7 7 -2 8 8 4 . Cervantes E ., Rodriguez A. and Nicolas G. (1994), E th ylene regulates the expression of a cysteine proteinase gene during germination of chickpea ( Cicer arietinum L .). Plant Mol. Biol. 25, 2 0 7 -2 1 5 . Chang M. M., Hadwiger L. A. and Horovitz D. (1992), M olecular characterizarion of a pea ß-l,3-glucanase induced by Fusarium solani and chitosan challenge. Plant Mol. Biol. 20, 6 0 9 -6 1 8 . Chomczynski P. (1992), One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal. Biochem. 201, 1 3 4 -1 3 9 Chomczynski P. and Sacchi N. (1987), Single-step method of RN A isolation by acid guanidinium thiocyanate-phe...

Sequence analyses of eight metribuzin-resistant mutants of photoautotrophic Chenopodium rubrum cell cultures revealed new mutations in the psbA gene coding for the 32 kDa herbicide binding protein. Mutants were found to possess either two or three changes in the amino acid sequence of the Di-protein between positions 219 and 272.

The fatty acid compositions of plastidic and extra-plastidic membrane lipids of two metri-buzin-resistant cell lines L4 and L7 of C henopodium rubrum were determined after growth in the absence and in the presence of the herbicide and compared with those o f wild type cells. Fatty acid biosynthesis was markedly affected in all cell lines by metribuzin treatment. In the absence and in the presence of metribuzin alterations of the fatty acid com position of the various lipid classes were, as compared to wild type cells, generally lower in the highly resistant L4 cells than in the less resistant L7 cells. The two resistant cell lines demonstrated a higher degree of unsaturation within the plastidic monogalactosyldiacylglycerols (L4 cells also within plastidic digalactosyldiacylglycerols) and, particularly, within the predominantly extra-plastidic phosphatidylcholines (L7 cells also within the predominantly extra-plastidic phosphatidylethanolamines), whereas the degree of unsaturation was slightly altered in the plastidic phosphatidylglycerols. Within the two metribuzin-resistant cell lines, the highly resis­ tant L4 cells differed from the less resistant L7 cells by increased a-linolenic acid/palmitic acid ratios in both the plastidic and extra-plastidic membrane lipids suggesting that particu­ larly in L4 cells higher proportions of linolenate are formed as a result o f selection pressure. On the other hand, the proportion of linoleate was increased predominantly in extra-plastidic membrane lipids of both L4 and L7 cells which explains a raise in linoleic acid/palmitic acid ratios in both cell lines as compared to wild-type cells. Moreover, in the absence of metri­ buzin decreased proportions o f fram-3-hexadecenoic acid were found in phosphatidylglycer­ ols of L4 and, particularly, of L7 cells as compared to the wild type cells. It is suggested that L4 and L7 cells -having multiple mutations in the psbA gene as observed earlier -are additionally characterized by increased degree of unsaturation of acyl moieties in various polar lipids, e.g. linoleoyl moieties in L4 and L7 cells as well as linolenoyl moieties particularly in highly resistant L4 cells. This increase gives rise to a change in membrane fluidity and may finally lead to increased metribuzin resistance.

In eight metribuzin-resistant photoautotrophic cell cultures of Chenopodium rubrum (Thiemann and Barz, 1994 a, b) sequence analyses of a part of the psbA gene coding for the photosystem -II D1 protein had revealed different double and triple mutations within the herbicide binding niche of the protein (Schwenger-Erger et al., 1993). Two pairs of the exam­ined cell lines carried identical mutations within this part of the protein, although their growth performance and their herbicide resistance patterns were different, both at the cellu­lar and the thylakoid level. Starting from the known part of the psbA gene we have amplified the remaining psbA sequences using inverse polymerase chain reaction. Thus the complete sequence of the coding part of the gene was elucidated. After sequence analyses we found an additional amino acid exchange at the position 184 (ile → asn) of the D1 protein in cell line L1. Metabolic consequences of this mutation are discussed. Partial sequence analyses of the psbD gene of the herbicide resistant cell culture lines revealed no mutation within that part o f the D2 protein, which is in direct contact with the D1 protein.

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