Senescence-associated down-regulation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase delays harvest-induced senescence in broccoli.

To gain an in-depth understanding of the role of ethylene in post harvest senescence, we used broccoli (Brassica oleracea var. italica) as our model species. The senescence-associated asparagine synthetase (AS) promoter from asparagus was used to drive the expression of an antisense 1-aminocyclopropane-1-carboxylate oxidase (ACO) cDNA from broccoli, BoACO2, to reduce ethylene production following harvest. Physiological analyses revealed that transgenic broccoli lines harbouring the antisense BoACO2 gene construct (designated as AS-asACO) displayed delayed senescence in both detached leaves and detached heads as measured by hue angle. Harvested floret tissue from these plants also showed a delayed loss of chlorophyll, lower protease activity and higher total protein content, and changes in transcript levels of senescence marker genes when compared with wild type and transgenic lines transformed with an empty T-DNA. Genes that were down-regulated included those coding for cysteine protease (BoCP5), metallothionein-like protein (BoMT1), hexokinase (BoHK1), invertase (BoINV1) and sucrose transporters (BoSUC1 and BoSUC2). Northern analysis for BoACO1 and BoACO2, ACO assays and western analysis, revealed reduced ACO transcript, enzyme activity and protein accumulation, as well as reduced ethylene production in the transgenic AS-asACO lines when compared with controls, confirming that a key enzyme regulating ethylene biosynthesis was reduced in these plants. This, together with the changes observed in gene expression, confirm a significant role for ethylene in regulating the events leading to senescence in broccoli following harvest.

Analysis of the asparagus (Asparagus officinalis) asparagine synthetase gene promoter identifies evolutionarily conserved cis-regulatory elements that mediate Suc-repression.

In asparagus (Asparagus officinalis L.), increased levels of asparagine (Asn) and Asn synthetase (AS) transcript are detected during foliar senescence and in harvested spears, possibly triggered by signals from a reduced supply of carbohydrate. To identify cis-elements mediating this regulation, the asparagus AS gene promoter was isolated and analysed by DNA sequencing, followed by expression of AS::GUS (ß-glucuronidase) reporter-gene constructs in transgenic tissue, and electrophoretic mobility shift assays (EMSA). The 1958-base pair (bp) region of the AS promoter upstream of the translation initiation ATG (-1958 bp region) was sufficient to confer sucrose (Suc)-regulated expression on the GUS reporter gene in asparagus callus and protoplasts, which were transformed by particle bombardment and electroporation, respectively. Removal of Suc from callus or protoplast media resulted in the induction of GUS activity. Deletion analysis of this 1958-bp fragment identified elements in the -640 to -266bp region as important for both high GUS levels and mediating the Suc response. This was supported by EMSA results, which showed the formation of three nuclear protein-DNA complexes with the -558 to -284 bp fragment of the promoter. A 20-bp oligonucleotide, designed to match the sequence from -423 to -404 bp, was able to out-compete formation of one of these protein-DNA complexes, suggesting a specific interaction with this sequence. This region of the promoter, overlapping with the 20-bp oligonucleotide sequence, contains a 10-bp stretch identical to a sequence previously shown to mediate low Suc induction of an Oryza sativa (rice) α-amylase gene, and may thus represent a conserved Suc-responsive element.

Distinct cis-elements in the Asparagus officinalis asparagine synthetase promoter respond to carbohydrate and senescence signals.

The Asparagus officinalis L. asparagine (Asn) synthetase (AS) promoter was analysed for elements responding to carbohydrate and senescence signals. Transgenic Arabidopsis thaliana L. plants containing deletion constructs of the -1958 bp AS promoter linked to the ß-glucuronidase (GUS) reporter gene (AS::GUS) were analysed by measuring GUS specific activity. Inclusion of sucrose (Suc), glucose (Glc) or fructose (Fru) in plant media repressed levels of GUS activity in -1958AS::GUS plants, regardless of the light environment, with increases in GUS found 1 d after incubation on Suc-lacking media. Hexokinase is likely to be involved in the signal pathway, as Suc, Glc, Fru, 2-deoxy-d-glucose and mannose were more effective repressors than 3-O-methylglucose, and the hexokinase inhibitor mannoheptulose reduced repression. Plants containing AS::GUS constructs with deletions that reduced the promoter to less than -405 bp did not show low sugar induction. AS::GUS activity was significantly higher in excised leaves induced to senesce by dark storage for 24 h, compared to fresh leaves, for lines containing at least -640 bp of the AS promoter but not those with -523 bp or smaller promoter fragments. Fusion of the -640 to -523 bp region to a -381AS::GUS construct generated a promoter that retained senescence induction but lacked low sugar induction. Alignment of this region to the 33-bp senescence-related sequence of the Arabidopsis and Brassica napus L. SAG12 promoters identified the sequence TTGCACG as being conserved in all the promoters, and which may be an important senescence-responsive element.

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

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.

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

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.

Controlled atmospheres and sugar can delay malate synthase gene expression during asparagus senescence.

A cDNA clone encoding malate synthase (MS; EC 4.1.3.2) was isolated from a 48-h postharvest asparagus (Asparagus officinalis L.) spear cDNA library using a MS clone from Brassica napus. The asparagus MS (AoMS1) cDNA hybridized to a 1.9-kb transcript that increased in abundance preferentially in spear-tip tissue during postharvest storage. The AoMS1 transcript also accumulated during natural foliar senescence of asparagus fern. The cDNA consists of 1960 nucleotides with an open reading frame of 1665 nucleotides or 555 amino acids, and encodes a deduced protein with a predicted Mr of 63 kDa and a pI of 8.1. The deduced amino acid sequence of AoMS1 showed high identity with the B. napus MS clone (77.2%) used to isolate it, and with MS from cucumber (77%). Genomic Southern analysis suggests that a single gene in asparagus encodes AoMS1. Controlled- atmosphere treatments aimed at reducing deterioration of harvested asparagus spears reduced the expression of AoMS1. The reduction was correlated with the reduced oxygen level, and reduced MS enzyme activity was also observed. Asparagus cell cultures were used to test the role of sugar status in regulating AoMS1 gene expression. In cultures without sucrose there was an accumulation of AoMS1 transcript that was absent in cultures containing sucrose.