Tài liệu Báo cáo Y học: Divergent members of a soybean (Glycine max L.) 4-coumarate:coenzyme A

Divergent members of a soybean (Glycine max L.)

4-coumarate:coenzyme A ligase gene family

Primary structures, catalytic properties, and differential expression

Christian Lindermayr1

, Britta Mo¨ llers1

, Judith Fliegmann1

, Annette Uhlmann1

, Friedrich Lottspeich2

,

Harald Meimberg3 and Ju¨ rgen Ebel1

1

Botanisches Institut der Universita¨t, Mu¨nchen, Germany; 2

Max-Planck-Institut fu¨r Biochemie, Martinsried, Germany; 3

Institut fu¨r Systematische Botanik der Universita¨t, Mu¨nchen, Germany

4-Coumarate:CoA ligase (4CL) is involved in the formation

of coenzyme A thioesters of hydroxycinnamic acids that are

central substrates for subsequent condensation, reduction,

and transfer reactions in the biosynthesis of plant phenyl￾propanoids. Previous studies of 4CL appear to suggest that

many isoenzymes are functionally equivalent in supplying

substrates to various subsequent branches of phenylpropa￾noid biosyntheses. In contrast, divergent members of a 4CL

gene family were identified in soybean (Glycine max L.). We

isolated three structurally and functionally distinct 4CL

cDNAs encoding 4CL1, 4CL2, and 4CL3 and the gene

Gm4CL3. A fourth cDNA encoding 4CL4 had high simi￾larity with 4CL3. The recombinant proteins expressed in

Escherichia coli possessed highly divergent catalytic effi￾ciency with various hydroxycinnamic acids. Remarkably,

one isoenzyme (4CL1) was able to convert sinapate; thus the

first cDNA encoding a 4CL that accepts highly substituted

cinnamic acids is available for further studies on branches of

phenylpropanoid metabolism that probably lead to the

precursors of lignin. Surprisingly, the activity levels of the

four isoenzymes and steady-state levels of their transcripts

were differently affected after elicitor treatment of soybean

cell cultures with a b-glucan elicitor of Phytophthora sojae,

revealing the down-regulation of 4CL1 vs. up-regulation of

4CL3/4. A similar regulation of the transcript levels of the

different 4CL isoforms was observed in soybean seedlings

after infection with Phytophthora sojae zoospores. Thus,

partitioning of cinnamic acid building units between

phenylpropanoid branch pathways in soybean could be

regulated at the level of catalytic specificity and the level of

expression of the 4CL isoenzymes.

Keywords: 4-coumarate:CoA ligase; differential regulation;

heterologous expression; plant defence; soybean (Glycine

max L.).

Phenylpropanoid compounds are major constituents of

higher plants. They can serve as flower pigments, UV

protectants, defence chemicals, signalling compounds, allelo￾pathic agents, and as building units of the phenolic support

polymer, lignin. Their synthesis is regulated both by

developmental processes and by environmental cues and it

proceeds via the general phenylpropanoid pathway and

subsequent specialized branches of phenylpropanoid meta￾bolism. Central to many of the biosynthetic pathways is the

activation of differently substituted cinnamic acids to the

corresponding CoA thioesters. This reaction is catalyzed by

4-coumarate:CoA ligase (4CL; EC 6.2.1.12), a member of

general phenylpropanoid metabolism.

The central position of 4CL, linking the general with

specialized branches of phenylpropanoid metabolism, led to

the suggestion that 4CL could play a pivotal role in

regulating the flux of the activated CoA ester intermediates

into subsequent biosynthetic pathways. This idea was

substantiated by the observation that isoenzymes of 4CL

in soybean (Glycine max), petunia (Petunia hybrida), pea

(Pisum sativum), oat (Avena sativa), and poplar (Populus ·

euramericana) displayed different substrate affinities and/or

tissue distribution [1–5]. In contrast, other plants apparently

contain only a single 4CL isoenzyme or isoforms that

exhibit similar substrate specificities [6–10]. In these cases,

the ring-modifications on the cinnamic acid derivatives,

which precede the partitioning into different pathways, may

proceed at the level of the activated esters, as well as the

aldehydes and alcohols, as proposed recently [11–14].

Therefore, the physiological relevance of the occurrence of

multiple 4CL in the former plants remains largely unknown.

4CL genes have been studied in a large variety of plants,

where they comprise small gene families in most cases. In a

number of plants, including parsley (Petroselinum crispum),

loblolly pine (Pinus taeda), and potato (Solanum tuberosum),

the genes encode identical or very similar proteins [7,15,16],

whereas in other plants, such as tobacco (Nicotiana

Correspondence to J. Ebel, Botanisches Institut der Universita¨t

Mu¨nchen, Menzinger Strasse 67, D-80638 Mu¨nchen, Germany.

E-mail: [email protected]

Abbreviation: 4CL, 4-Coumarate:coenzyme A ligase.

Enzyme: 4-coumarate:CoA ligase (EC 6.2.1.12).

Note: the nucleotide sequence data reported were deposited under

GenBank accession nos AF279267 for Gm4CL1 cDNA, AF002259 for

4CL14 (Gm4CL2 cDNA), AF002258 for 4CL13 (Gm4CL3 genomic),

and X69955 for 4CL16 (Gm4CL4 cDNA).

(Received 23 October 2001, revised 28 December 2001, accepted

9 January 2002)

Eur. J. Biochem. 269, 1304–1315 (2002) Ó FEBS 2002