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

Abstract

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.

Stress Responses in Alfalfa (XXI. Activation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase Genes Does Not Contribute to Changes in Metabolite Accumulation in Elicitor-Treated Cell-Suspension Cultures)

“…The PAL probe used for northern blot analysis and detection of in vitro transcripts was the 0.47-kb interna1 HindIII fragment of the alfalfa PAPAL1 cDNA clone (Gowri et al, 1991b). The alfalfa COMT probe was the 1.3-kb insert from plasmid pCOMTl (Gowri et al, 1991a).…”

“…; Gowri et al, 1991aGowri et al, , 1991b). We observed a similar level of PAL induction to that reported here with no accompanying increase in COMT activity in other alfalfa cell lines (data not shown).…”

Spatial patterns of expression of flavonoid/isoflavonoid pathway genes during interactions between roots of Medicago truncatula and the mycorrhizal fungus Glomus versiforme

“…The plasmids pAPAL1 (Gowri et al, 1991 ), pCHS1 (Junghans et al, 1993), plFRalfl (Paiva et al, 1991, pCHM'I'4 and pCHI (Maxwell eta/., 1993) are all derived from pBluescript and contain a polylinker flanked by the T3 and T7 promoters. Plasmid DNA from each of these clones was linearized and used as a template for the in vitro synthesis of 3sS-labelled antisense and sense RNA probes.…”

Stress Responses in Alfalfa (Medicago sativa L.) (XIV. Changes in the Levels of Phenylpropanoid Pathway Intermediates in Relation to Regulation of L-Phenylalanine Ammonia-Lyase in Elicitor-Treated Cell-Suspension Cultures)

“…A cDNA complementary to an alfalfa PAL (471-bp HindIII interna1 coding region fragment [positions 831-1301]) (Gowri et al, 1991), a 1.6-kb full-length alfalfa CHS cDNA (K. Dalkin, H. Junghans, and R.A. Dixon, unpublished results), and the cDNA clone for the constitutively expressed H1 transcript from bean (Lawton and Lamb, 1987) were used in northem blot analyses as described below. The PAL and CHS probes do not discriminate between different members of the alfalfa pal and chs multigene families.…”

“…CHS catalyzes the conversion of 4-coumaroyl Co-A into 2',4,4'-trihydroxychalcone, the first branch point into the production of flavonoids and isoflavonoids. The phytoalexin defense response involves the rapid transcriptional activation of genes encoding a number of phytoalexin biosynthetic enzymes, including PAL and CHS (Hahlbrock and Scheel, 1989;Dixon and Hamson, 1991;Gowri et al, 1991;. A large body of evidence suggests that phenylpropanoid pathway intermediates can regulate expression of the pathway at the posttranslational and transcriptional levels.…”