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Journal of Experimental Botany, Vol. 54, No. 384, pp. 1045-1056, March 1, 2003
© 2003 Oxford University Press

Identification of dehydration-responsive cysteine proteases during post-harvest senescence of broccoli florets

Received 24 September 2002; Accepted 26 November 2002

Simon A. Coupe2,, Ben K. Sinclair, Lyn M. Watson, Julian A. Heyes and Jocelyn R. Eason3,

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

1 The nucleotide sequence data reported here appears in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under accession numbers: AF454956 (BoCP1), AF454957 (BoCP2), AF454958 (BoCP3), and AF454959 (BoCP4).
2 Present address: Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
3To whom correspondence should be addressed. Fax: +64 6 351 7050. E-mail: easonj{at}crop.cri.nz
Abbreviations: CA, Controlled atmosphere; MRI, magnetic resonance imaging.

Harvest-induced senescence of broccoli results in tissue wilting and sepal chlorosis. As senescence progresses, chlorophyll and protein levels in floret tissues decline and endo-protease activity (measured with azo-casein) increases. Protease activity increased from 24 h after harvest for tissues held in air at 20 °C. Activity was lower in floret tissues from branchlets that had been held in solutions of sucrose (2% w/v) or under high carbon dioxide, low oxygen (10% CO2, 5% O2) conditions. Four protease-active protein bands were identified in senescing floret tissue by zymography, and the use of chemical inhibitors of protease action suggests that some 44% of protease activity in senescing floret tissue 72 h after harvest is due to the action of cysteine and serine proteases. Four putative cysteine protease cDNAs have been isolated from broccoli floret tissue (BoCP1, BoCP2, BoCP3, BoCP4). The cDNAs are most similar (73–89% at the amino acid level) to dehydration-responsive cysteine proteases previously isolated from Arabidopsis thaliana (RD19, RD21). The mRNAs encoded by the broccoli cDNAs are expressed in floret tissue during harvest-induced senescence with mRNA accumulating within 6 h of harvest for BoCP1, 12 h of harvest for BoCP4 and within 24 h of harvest for BoCP2 and BoCP3. Induction of the cDNAs is differentially delayed when broccoli branchlets are held in solutions of water or sucrose. In addition, the expression of BoCP1 and BoCP3 is inhibited in tissue held in atmospheres of high carbon dioxide/low oxygen (10% CO2, 5% O2). The putative cysteine protease mRNAs are expressed before measurable increases in endo-protease activity, loss of protein, chlorophyll or tissue chlorosis.

Key words: Brassica oleracea, broccoli, controlled atmosphere, post-harvest, senescence, wilting.


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