We report on the production and selection of transgenic Brassica oleracea var. Italica lines with a downregulated soluble acid invertase (BoINV2). Explants of broccoli (cv. Triathlon) were transformed with an antisense construct of BoINV2 under the control of an Asparagus officinalis‐derived harvest‐induced promoter using Agrobacterium tumefaciens‐mediated transformation. BoINV2 is upregulated in wild‐type broccoli floret tissue after harvest. Transgenic broccoli lines showed reduced BoINV2 mRNA accumulation immediately after harvest compared with wild‐type. Downregulation of BoINV2 had no significant impact on the expression of a second broccoli acid invertase gene (BoINV1), but plants with downregulated BoINV2 also had lower expression of a senescence‐associated cysteine protease (BoCP5) compared with wild‐type. The total soluble sugar levels in floret tissue of antisense BoINV2 lines were greater than wild‐type tissue after harvest (up to 1.5 times higher). Soluble protein content of wild‐type tissue decreased from 48 h after harvest with an increase in protease activity. In comparison, two antisense BoINV2 lines retained at‐harvest levels of soluble protein until 72 and 96 h after harvest and had lower postharvest endoprotease activity compared with wild‐type. Antisense BoINV2 lines also had a slower rate of floret sepal chlorosis after harvest compared with wild‐type.

The current study examines the transcription of four genes (BoCAR1A, BoCAR5, BoCAR6-4 and BoCAR25) found to be up-regulated in response to high CO(2)/low O(2) treatment in broccoli (Brassica oleracea). Messenger RNA levels for the four genes declined after tissues were removed from CA. Physiological and biochemical changes and gene expression patterns were examined in broccoli tissues held in one of four different atmospheres, namely air (<1% CO(2), 21% O(2)), high carbon dioxide and low oxygen (CA 10% CO(2), 5% O(2)), low oxygen (0% CO(2), 5% O(2)), and high carbon dioxide (10% CO(2), 20% O(2)). In a second trial gene expression was examined in tissues held for short periods in CA (6h, 12h or 24h) followed by air. Broccoli tissues were also exposed to CA after 48 h in air to determine whether CA treatment was effective in up-regulating the CA-responsive genes and/or delaying senescence after early senescence-associated gene changes had been initiated. Northern analysis showed that a combined high CO(2) and low O(2) atmosphere was more effective than high CO(2) or low O(2) alone for inducing maximum gene expression and delaying postharvest broccoli senescence. In addition, broccoli tissues responded to CA treatment after a 48-h period in air with increased CA-responsive gene expression. Certain transcripts were down-regulated in tissues exposed to salt and water stresses that promoted senescence, and down-regulated in tissues treated with cytokinin, a treatment that delays postharvest senescence in broccoli. The up-regulation of these four BoCAR genes appears to be specific to CA treatment in harvested broccoli tissues.

Broccoli (Brassica oleracea L.) tissue held in a controlled atmosphere (CA; 10% carbon dioxide and 5% oxygen) senesces more slowly than tissue held in air. CA-treated broccoli tissues lose less water and soluble sugars, have lower protease activity, and have no significant loss of color (hue angle, chlorophyll content) for 96h after harvest (20°C, dark) compared to tissue held in air that starts to senesce and yellow after 48h. The current study examined differential gene expression in broccoli tissues in response to postharvest CA treatment. This genetic analysis was undertaken to identify CA-responsive genes that may act as signaling elements and repress postharvest senescence processes. CA-responsive genes with up- and down-regulated expression (compared to air controls) were isolated after a 6h CA treatment by differential display-polymerase chain reaction. The candidate CA-responsive genes included a number of novel genes without previously assigned functions, and genes of known function previously found to be regulated by stress (e.g. dehydration, salt stress, low temperature, and sugar starvation).

An aleurain-like protein, BoCP5, is up-regulated during harvest-induced senescence in broccoli floret and leaf tissue. BoCP5 is most closely related to an Arabidopsis protein (91%, AAF43041) and has 71% identity to barley aleurain (P05167). The mRNA for this gene accumulates within 6 h after harvest in broccoli florets, and its expression is reduced in tissue that has been held in senescence-delaying treatments (e.g. water, sucrose feeding, controlled atmosphere). The gene is also expressed in leaves during aging-related and harvest-induced senescence. Analysis of protein bands that cross-react with antibodies raised to the bacterial BoCP5 fusion protein, revealed prominent immunoreactive bands at ca. 26, 28, 31, and 38 kD in floret tissue. The 31 kD band was absent in protein extracts from leaf tissue. Agrobacterium-mediated transformation was used to produce transgenic broccoli plants with down-regulated BoCP5. A reduction in the postharvest expression of BoCP5 in floret tissue was achieved for four transgenic lines in the current study. In three of these lines postharvest floret senescence (yellowing) was delayed, and florets contained significantly greater chlorophyll levels during postharvest storage at 20 °C than wild-type plants. Line 4 showed the greatest down-regulation of BoCP5, and in this line postharvest protease activity remained at pre-harvest levels, and the yield of soluble proteins extracted from florets after harvest was significantly greater than that of wild-type tissue.

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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 β-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.

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.

Cysteine protease inhibitors delayed the senescence of Sandersonia aurantiaca Hook. flowers. Tepal fading and wilting occurred later in the 2,2'-dipyridyl-treated flowers, and these flowers had a greater soluble protein content and less active endoproteases compared with control flowers that were held in water. Biochemical analysis revealed the presence of several protease-active bands in the soluble protein fraction of Sandersonia tepals. Activity of the polypeptides increased as flower senescence progressed. Western analysis with an antibody raised against the castor bean cysteine proteinase identified homologous proteins in Sandersonia flowers (ca 46, 41 and 31 kDa). Three cDNAs encoding cysteine proteinases were isolated from Sandersonia tepals (PRT5, PRT15 and PRT22). Expression of all three increased in tepals as senescence progressed. mRNAs for PRT5 were detected only in senescing flower tissue, whereas PRT15 and PRT22 were expressed in leaf, stem and root tissue. PRT5 has significant homology to C-terminus KDEL proteins, which have a role in the degradation of plant cell contents during programmed cell death. PRT15 is most similar to cysteine proteinases with a long C-terminal extension, whereas PRT22 is homologous to stress-induced cysteine proteinases.