Tài liệu Báo cáo khoa học: Biochemical and molecular characterization of hazelnut (Corylus avellana)

Biochemical and molecular characterization of hazelnut

(Corylus avellana) seed lipoxygenases

Angelo Santino1

, Angelo De Paolis1

, Antonia Gallo1,*, Angela Quarta1

, Rod Casey2 and Giovanni Mita1

1

Institute of Sciences of Food Production C.N.R. Section of Lecce, Italy; 2

John Innes Centre, Norwich, UK

Plant lipoxygenases (LOXs) are a class of dioxygenases

which display diverse functions in several physiological

processes such as growth, development and response to

biotic and abiotic stresses.Even though LOXs have been

characterized from several plant species, the physiological

role of seed LOXs is still unclear.With the aim to better

clarify the occurrence of LOXs and their influence on

hazelnut seed quality, we carried out the biochemical and

molecular characterization of the main LOX isoforms

expressed during seed development.A genomic clone con￾taining a complete LOX gene was isolated and fully char￾acterized.The 9887 bp sequence reported contains an open

reading frame of 5334 bp encoding a putative polypeptide of

99 kDa.Semiquantitative RT-PCR carried out from RNAs

extracted from seeds at different maturation stages showed

that LOXs are mainly expressed at early developmental

stages.These results were confirmed by LOX activity assays.

Biochemical characterization of the reaction products of the

hazelnut LOX indicated that it is a 9-LOX.Two cDNAs

were isolated by RT-PCR carried out on total RNA from

immature hazelnut seeds.Sequence analysis indicated that

the two cDNAs are highly homologous (91.9% degree of

identity) and one of these corresponded exactly to the

genomic clone.The deduced amino acid sequences of the

hazelnut LOXs showed that they are closely related to

a previously reported almond LOX (79.5% identity) and, to

a lesser extent, to some LOXs involved in plant responses

to pathogens (cotton and tobacco LOXs, 75.5 and 74.6%

identity, respectively).The physiological role of hazelnut

LOXs and their role in influencing seed quality are also

discussed.

Keywords: Corylus avellana; hazelnut; lipoxygenases; seed

quality.

Lipoxygenases (LOX, EC 1.13.11.12) are a class of

widespread dioxygenases that catalyze the addition of

oxygen to polyunsaturated fatty acids containing a cis,cis￾1,4-pentadiene structure.The hydroperoxides produced by

the LOX reaction are the starting point for a series of

other enzymatic reactions which lead to the synthesis of a

group of biologically active compounds collectively named

oxylipins.In animal cells, the LOX pathway has been

extensively studied as it is responsible for the synthesis of

leukotrienes and lipoxins belonging to the eicosanoid

family which are involved in many physiological processes

[1].In plants the biosynthesis of phytooxylipins is initi￾ated by the oxygen insertion at C-9 or C-13 of linoleic

(C18 : 2) or linolenic acids (C18 : 3).For this reason plant

LOXs are commonly referred as 9-LOXs or 13-LOXs.

Both 9- or 13-hydroperoxides are further converted to

different compounds through the action of the other

enzymes belonging to different branches of the LOX

pathway (hydroperoxide lyase, allene oxide synthase,

divinyl ether synthase, reductase, peroxygenase).At the

end of these enzymatic reactions the molecules synthesized

display a wide variety of physiological roles in plant

development and response to biotic and abiotic stresses.In

this context lipoxygenases, providing the substrates for the

activity of all the enzymes located downstream on the

pathway, have a primary role in the biosynthesis of

phytooxylipins and can affect their availability inside the

plant cell.The biological role of specific LOX isoforms

has been recently clarified by an antisense approach and

their depletion was able to influence plant development or

pest/pathogen resistance [2,3].Transgenic potato plants

depleted in the expression of a specific 9-LOX showed an

abnormal tuber development [4].Furthermore Arabidopsis

and potato transgenic plants with diminished levels of

specific chloroplastic LOXs have reduced levels of wound

inducible mRNAs [5,6].

LOXs are widespread in the seeds of many plant species;

in some cases such as soybean and other legumes they are

abundant proteins and might also function as storage,

rather than defence, proteins.This hypothesis is supported

Correspondence to A.Santino, ISPA-CNR, via Monteroni,

73100 Lecce, Italy.Fax:/Tel.: + 39 0832 420000,

E-mail: [email protected]

Abbreviations: 9-HODE, (9S,10E,12Z)-9-hydroxy-10,12-octadeca￾dienoic acid; 13-HODE, (13S,9Z,11E)-13-hydroxy-9,11-octadeca￾dienoic acid; LOX, lipoxygenase; RP-HPLC, reverse-phase HPLC;

SP-HPLC, straight-phase HPLC.

Enzyme: lipoxygenase (EC 1.13.11.12).

Note: a web site is available at http://www.ispa.cnr.it/

Note: The nucleotide sequence reported is in the EMBL database

under the accession number AJ417975.

*Present address: Institute of Sciences of Food Production C.N.R.

Bari, Italy.

(Received 18 July 2003, revised 2 September 2003,

accepted 12 September 2003)

Eur. J. Biochem. 270, 4365–4375 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03831.x