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Journal of Experimental Botany, Vol. 55, No. 394, pp. 59-68, January 1, 2004
© 2004 Oxford University Press

Cell and Molecular Biology, Biochemistry and Molecular Physiology

Molecular analysis of programmed cell death during senescence in Arabidopsis thaliana and Brassica oleracea: cloning broccoli LSD1, Bax inhibitor and serine palmitoyltransferase homologues

Received 6 February 2003; Accepted 3 October 2003

Simon A. Coupe1,*, Lyn M. Watson1, Dacey J. Ryan1, Tatyana T. Pinkney1 and Jocelyn R. Eason1,{dagger}

1 The New Zealand Institute for Crop and Food Research Limited, Food Industry Science Centre, Private Bag 11 600, Palmerston North, New Zealand

* Present address: Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
{dagger} To whom correspondence should be sent. Fax: +64 6 351 7050. E-mail: easonj{at}crop.cri.nz
DNA sequences: The nucleotide sequence data reported will appear in the GenBank Nucleotide Sequence Databases under accession numbers: AF453320 (BoBI-1), AF453321 (BoBI-2), AF453322 (BoLSD1), AF453323 (BoBLSD2), AF525281 (BoSPT1), AF525282 (BoSPT2), and AF513990 (18S rRNA).
Abbreviations: BI, Bax inhibitor; DALE, days after leaf emergence; DAPI, 4,6-diamidino-2-phenylindole; HR, hypersensitive response; LSD1, lesion simulating disease; PCD, programmed cell death; SPT, serine palmitoyltransferase; TUNEL, TdT-mediated dUTP nick end labelling.

This study was undertaken to characterize the programmed cell death (PCD) processes that occur during detached and natural on-plant senescence and correlate them with the expression of putative regulatory genes that may be involved in the process. DNA fragmentation and TUNEL analysis of broccoli florets showed that DNA was processed into fragments of approximately 180 bp after 48 h of harvest-induced tissue senescence. Characteristic laddering patterns were also visible in Arabidopsis leaves undergoing natural on-plant senescence and during detached senescence. Several recently isolated plant proteins have been assigned a PCD role, for example, the zinc finger containing protein, LSD1 (lesion simulating disease); Bax inhibitor (BI); and serine palmitoyltransferase (SPT), an enzyme in the sphingolipid signalling pathway. Two cDNAs encoding each of these proteins were isolated from broccoli (BoBI-1, BoBI-2, BoLSD1, BoLSD2, BoSPT1, BoSPT2), and the mRNAs increased during harvest-induced senescence in floret tissue. Expression of the Arabidopsis homologues (AtBI-1, AtLSD1, AtSPT1) were also characterized during detached leaf senescence in Arabidopsis leaves. AtBI-1 expression was constitutively expressed during detached senescence, AtLSD1 expression remained constitutively low, and AtSPT1 expression increased during detached senescence.

Key words: Ceramide, DNA laddering, post-harvest, sphingolipid biosynthesis, TUNEL analysis.


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