The nitrate induction of NADH:nitrate reductase mRNA in maize roots, scutella and leaves was investigated in the presence and absence of inhibitors of protein synthesis. In the absence of inhibitors, nitrate treatment caused a fairly rapid (2 to 3 h) increase in the level of the nitrate reductase transcript in all tissues. When cytoplasmic protein synthesis was inhibited by cycloheximide, nitrate reductase mRNA was induced by nitrate in all tissues to levels equal to or greater than those found with nitrate treatment alone. Treatment of maize tissues with cycloheximide in the absence of nitrate had only a small effect on the accumulation of the nitrate reductase mRNA. Inhibition of organellar protein synthesis with chloramphenicol also had little or no effect on nitrate-induced nitrate reductase mRNA accumulation in roots and scutella, but did appear to partially inhibit appearance of transcript in leaves. Excision of scutella in the absence of nitrate was sufficient to cause some accumulation of the nitrate reductase transcript. Since cytoplasmic protein synthesis was not required for expression of nitrate reductase transcripts, induction of these transcripts by nitrate is a primary response of maize to this environmental signal. Thus, it appears that the signal transduction system mediating this response is constitutively expressed in roots, scutella and leaves of maize.

S-Adenosyl-L-methionine:caffeic acid 3-0-methyltransferase (COMT, EC 2.1.1.6) catalyzes the conversion of caffeic acid to ferulic acid, a key step in the biosynthesis of lignin monomers. We have isolated a functionally active cDNA clone (pCOMT1) encoding alfalfa (Medicago sativa L.) COMT by immunoscreening a XZAPII cDNA expression library with anti-(aspen COMT) antibodies. The derived amino acid sequence of pCOMT1 is 86% identical to that of COMT from aspen. Southern blot analysis indicates that COMT in alfalfa is encoded by at least two genes. Addition of an elicitor preparation from bakers' yeast to alfalfa cell suspension cultures resulted in a rapid accumulation of COMT transcripts, which reached a maximum level around 19 hours postelicitation. Northern blot analysis of total RNA from different organs of alfalfa plants at various developmental stages showed that COMT transcripts are most abundant in roots and stems. Transcripts encoding ATP: i-methionine-S-adenosyl transferase (AdoMet synthetase, EC 2.5.1.6), the enzyme responsible for the synthesis of the methyl donor for the COMT reaction, were coinduced with COMT transcripts in elicitor-treated cells and exhibited a similar pattern of expression to that of COMT in different organs of alfalfa plants at various stages of development.

In etiolated squash (Cucurbita maxima L.) cotyledons, nitrate-inducible NADH:nitrate reductase activity and protein were increased in darkness by red light pulses with red/far-red photoreversibility. Continuous far-red light also led to increased levels of nitrate reductase activity and protein. Poly(A)+ RNA, which hybridizes to squash nitrate reductase cDNA, was also increased by light treatments. Thus, we found that after nitrate triggering, nitrate reductase expression appears to be regulated by light via phytochrome.

An expression library containing cDNAs derived from transcripts from fungal elicitor-treated alfalfa cell suspension cultures was screened with an antiserum raised against phenylalanine ammonia-lyase (PAL) from alfalfa. A single immunoreactive clone was isolated which encoded a full-length PAL cDNA (APAL1) consisting of a 2175 bp open reading frame, 96 bp 5'-untranslated leader and 128 bp 3'-non-coding region. The deduced amino acid sequence was 86.5% similar to that of the PAL2 gene of bean, and encoded a polypeptide of Mr 78,865. A second PAL cDNA species was isolated, whose 3'-untranslated region was 86% identical to that of APAL1. Southern blot analysis indicated that PAL is encoded by a small multigene family in alfalfa. PAL transcript levels were rapidly and massively induced, and preceded increased PAL extractable activity, on exposure of alfalfa suspension cells to elicitor from baker's yeast. PAL transcripts were most abundant in roots, stems and petioles during growth and development of alfalfa seedlings. These studies provide the basis for an examination of the developmental and environmental control of a key enzyme of phenylpropanoid synthesis in a plant species which is readily amenable to stable genetic transformation.

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