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

Abstract

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 degrees 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% CO(2), 5% O(2)) 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% CO(2), 5% O(2)). The putative cysteine protease mRNAs are expressed before measurable increases in endo-protease activity, loss of protein, chlorophyll or tissue chlorosis.

Gene expression in opening and senescing petals of morning glory (Ipomoea nil) flowers

“…In15, a putative cysteine protease, showed similarity with PRT22 related to tepal senescence of Sandersonia (Eason et al 2002). In21, a putative cysteine protease with a granulin domain, was similar to several genes related to senescence, i.e., BoCP2 of broccoli (Coupe et al 2003), ALSCYP1 of Alstroemeria petals (Wagstaff et al 2002), SEN102 of daylily petals (Guerrero et al 1998), and DCCP1 of carnation petals (Jones et al 1995). The sequence of the putative cysteine protease In33 was similar to that of genes related to leaf senescence, i.e., SPG31 of sweet potato (Chen et al 2002), SAG12 of Arabidopsis (Noh and Amasino 1999a), and BnSAG12 of Brassica (Noh and Amasino 1999b).…”

Effect of time of day for harvest and postharvest treatments on the sugar metabolism of broccoli (Brassica oleracea var. italica)

“…Postharvest treatments that delay the senescence process generally decrease the respiratory activity (Tian et al 1997, Funamoto et al 2002, Coupe et al 2003b) and allow the product to maintain a higher level of sugars. In our study, the content of fructose, glucose and sucrose decreased towards day 4 in all samples, as it was described in other cases, too (King and Morris 1994, Downs et al 1997, Pramanik et al 2005, Baclayon and Matsui 2009.…”

“…The two methods more widely used are cooling (Rodrigues and Rosa 1999) and modified atmosphere (MA) (Makhlouf et al 1989, Barth et al1993). Treatment with controlled or modified atmosphere substantially reduces senescence, delaying chlorophyll degradation and decreasing respiratory activity (Barth et al 1993, Coupe et al 2003b. Also, it has been used 1-methylcyclopropene (1-MCP), a substance that blocks ethylene receptors, as post-harvest treatment (Fan and Mattheis 2000).…”

Expression of Sweet Potato Senescence-Associated Cysteine Proteases Affect Seed and Silique Development and Stress Tolerance in Transgenic Arabidopsis

“…In broccoli, the florets showed water loss during post-harvest storage. Gene expression of papain-like cysteine proteases BoCP4, which exhibited high amino acid sequence identity with Arabidopsis RD19, was also found to be dehydration-responsive and was repressed by water and sucrose (Coupe et al, 2003).…”