Abstract
We have used high-resolution gas chromatography to determine the levels of trans-cinnamic acid (CA) and trans-4-coumaric acid (4CA) in alfalfa (Medicago sativa L.) cell-suspension cultures to address the role of these phenylpropanoid pathway intermediates as potential negative regulators of phenylalanine ammonia-lyase (PAL) in vivo. Exogenous addition of CA to elicitor-treated cultures resulted in rapid increases in endogenous CA, 4CA, and CA-conjugate levels associated with inhibition of the appearance of PAL transcripts. Treatment of elicited cultures with [alpha]-aminooxy-[beta]-phenylpropionic acid (AOPP), a potent and specific inhibitor of PAL activity in vivo, resulted in reductions of CA and 4CA, with concomitant increases in PAL transcripts and extractable enzyme activity. In contrast, treatment with tetcyclacis, an inhibitor of CA 4-hydroxylase, resulted in increased CA and CA-conjugate levels, decreased 4CA levels, and decreased PAL transcript levels and enzyme activity. In tetcyclasis-treated cells, the inhibition of PAL transcript appearance preceded the increase in the levels of free CA and its conjugates. In elicited cells in which the phenylpropanoid pathway was not perturbed by metabolic inhibitors, PAL transcripts accumulated rapidly and transiently, beginning to decline by 2 h postelicitation. Changes in levels of total free or conjugated CA or 4CA did not consistently correlate with these changes in transcript levels. We propose that regulation of PAL transcript levels by endogenous phenylpropanoid pathway intermediates could involve compartmentalized pools that may exist because of the microsomal localization of cinnamic acid 4-hydroxylase.
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- Bolwell G. P., Robbins M. P., Dixon R. A. Metabolic changes in elicitor-treated bean cells. Enzymic responses associated with rapid changes in cell wall components. Eur J Biochem. 1985 May 2;148(3):571–578. doi: 10.1111/j.1432-1033.1985.tb08878.x. [DOI] [PubMed] [Google Scholar]
- Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
- Dalkin K., Edwards R., Edington B., Dixon R. A. Stress Responses in Alfalfa (Medicago sativa L.): I. Induction of Phenylpropanoid Biosynthesis and Hydrolytic Enzymes in Elicitor-Treated Cell Suspension Cultures. Plant Physiol. 1990 Feb;92(2):440–446. doi: 10.1104/pp.92.2.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dixon R. A., Lamb C. J. Stimulation of de novo synthesis of L-phenylalanine ammonia-lyase in relation to phytoalexin accumulation in Colletotrichum lindemuthianum elicitor-treated cell suspension cultures of french bean (Phaseolus vulgaris). Biochim Biophys Acta. 1979 Sep 3;586(3):453–463. doi: 10.1016/0304-4165(79)90035-7. [DOI] [PubMed] [Google Scholar]
- Elkind Y., Edwards R., Mavandad M., Hedrick S. A., Ribak O., Dixon R. A., Lamb C. J. Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene. Proc Natl Acad Sci U S A. 1990 Nov;87(22):9057–9061. doi: 10.1073/pnas.87.22.9057. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gowri G., Paiva N. L., Dixon R. A. Stress responses in alfalfa (Medicago sativa L.) 12. Sequence analysis of phenylalanine ammonia-lyase (PAL) cDNA clones and appearance of PAL transcripts in elicitor-treated cell cultures and developing plants. Plant Mol Biol. 1991 Sep;17(3):415–429. doi: 10.1007/BF00040636. [DOI] [PubMed] [Google Scholar]
- KOUKOL J., CONN E. E. The metabolism of aromatic compounds in higher plants. IV. Purification and properties of the phenylalanine deaminase of Hordeum vulgare. J Biol Chem. 1961 Oct;236:2692–2698. [PubMed] [Google Scholar]
- Kessmann H., Edwards R., Geno P. W., Dixon R. A. Stress Responses in Alfalfa (Medicago sativa L.) : V. Constitutive and Elicitor-Induced Accumulation of Isoflavonoid Conjugates in Cell Suspension Cultures. Plant Physiol. 1990 Sep;94(1):227–232. doi: 10.1104/pp.94.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kroczek R. A., Siebert E. Optimization of northern analysis by vacuum-blotting, RNA-transfer visualization, and ultraviolet fixation. Anal Biochem. 1990 Jan;184(1):90–95. doi: 10.1016/0003-2697(90)90017-4. [DOI] [PubMed] [Google Scholar]
- Lamb C. J. Regulation of enzyme levels in phenylpropanoid biosynthesis: characterization of the modulation by light and pathway intermediates. Arch Biochem Biophys. 1979 Jan;192(1):311–317. doi: 10.1016/0003-9861(79)90097-3. [DOI] [PubMed] [Google Scholar]
- Lawton M. A., Lamb C. J. Transcriptional activation of plant defense genes by fungal elicitor, wounding, and infection. Mol Cell Biol. 1987 Jan;7(1):335–341. doi: 10.1128/mcb.7.1.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loake G. J., Choudhary A. D., Harrison M. J., Mavandad M., Lamb C. J., Dixon R. A. Phenylpropanoid pathway intermediates regulate transient expression of a chalcone synthase gene promoter. Plant Cell. 1991 Aug;3(8):829–840. doi: 10.1105/tpc.3.8.829. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mavandad M., Edwards R., Liang X., Lamb C. J., Dixon R. A. Effects of trans-Cinnamic Acid on Expression of the Bean Phenylalanine Ammonia-Lyase Gene Family. Plant Physiol. 1990 Oct;94(2):671–680. doi: 10.1104/pp.94.2.671. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Paiva N. L., Edwards R., Sun Y. J., Hrazdina G., Dixon R. A. Stress responses in alfalfa (Medicago sativa L.) 11. Molecular cloning and expression of alfalfa isoflavone reductase, a key enzyme of isoflavonoid phytoalexin biosynthesis. Plant Mol Biol. 1991 Oct;17(4):653–667. doi: 10.1007/BF00037051. [DOI] [PubMed] [Google Scholar]