Ben K. Sinclair

Regulation of Harvest-induced Senescence in Broccoli (Brassica oleracea var. italica) by Cytokinin, Ethylene, and Sucrose

Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. The two plant hormones ethylene and cytokinin are known to act antagonistically on harvest-induced senescence in broccoli: ethylene by accelerating the process, and cytokinin by delaying it. To determine the level at which these hormones influenced senescence, we isolated and monitored the expression of genes normally associated with senescence in broccoli florets treated with exogenous 6-benzyl aminopurine (6-BAP), 1-aminocyclopropane-1-carboxylic acid (ACC), a combination of 6-BAP and ACC, and sucrose, in the five days following harvest. Exogenous 6-BAP caused both a reduction (BoACO) and an increase (BoACS) in ethylene biosynthetic gene expression. The expression of genes used as senescence markers, BoCP5 and BoMT1, was reduced, whereas BoCAB1 levels were maintained after harvest in response to exogenous 6-BAP. In addition, the expression of genes encoding sucrose transporters (BoSUC1 and BoSUC2) and carbohydrate metabolizing enzymes (BoINV1 and BoHK1) was also reduced upon 6-BAP feeding. Interestingly, the addition of ACC prevented the 6-BAP-induced increase in expression of BoACS, but 6-BAP negated the ACC-induced increase in expression of BoACO. The culmination of these results indicates a significant role for cytokinin in the delay of senescence. The implication that cytokinin regulates postharvest senescence in broccoli by inhibiting ethylene perception and/or biosynthesis, thus regulating carbohydrate transport and metabolism, as well as senescence-associated gene expression, is discussed and a model presented.

Amino acid metabolism in senescing Sandersonia aurantiaca flowers: cloning and characterization of asparagine synthetase and glutamine synthetase cDNAs

We have studied nitrogen metabolism during senescence of the ethylene-insensitive flower Sandersonia aurantiaca Hook. We report here on the isolation and characterization of two cDNAs from senescing sandersonia tepals. SAND1 encodes asparagine synthetase (AS; EC 6.3.5.4) and SAND3 encodes glutamine synthetase (GS; EC 6.3.1.2). The accumulation of SAND3 mRNAs was consistently high throughout flower development and senescence and was not associated with senescence per se. SAND1 mRNAs started to accumulate in mature flowers just prior to visual signs of senescence. Sucrose feeding of individual flowers delayed tepal senescence and altered amino acid metabolism. The levels of Asn, Gln and Glu were higher and Asp levels were lower in the sucrose-fed flowers. The senescence-associated peak in Gln was delayed in sucrose-fed flowers compared to controls that were held in water. Sucrose feeding had no effect on the initiation of SAND1 transcription at the onset of flower senescence; however, the later decline in SAND1 mRNA abundance was delayed in sucrose-fed flowers. Elimination of GS activity and the subsequent reduction in Gln levels caused by phosphinothricin treat-ment was associated with a delay of SAND1 mRNA accumulation in senescing flowers.

Analysis of acid invertase gene expression during the senescence of broccoli florets

Broccoli (Brassica oleracea var. italica) kept at ambient temperatures after harvest senesce rapidly and sucrose levels decrease. However, if exogenous sucrose is supplied back to the plant, senescence symptoms can be delayed for up to 48 h. Acid invertase enzyme activity increases after harvest in broccoli stored at 20 °C and an investigation was undertaken to see if this enzyme has a role in the senescence process. Soluble acid invertase enzyme was extracted from fresh broccoli (0 h) and after 96 h of storage. There were two peaks of invertase activity, one at pH 5–5.5 and the other at pH 8. Broccoli stored in water and sucrose had reduced levels of acid invertase enzyme activity after 48 h compared to those stored dry. Similarly, broccoli kept in a controlled atmosphere (CA) of 10% CO2 and 5% O2 senesced at a slower rate and had significantly less acid invertase enzyme activity than air controls after 48 h. Two broccoli acid invertase cDNAs (BoINV1 and BoINV2) were isolated and these had 85 and 90% identity at the nucleotide and amino acid level. RNA blot analysis with BoINV1 and BoINV2 showed that transcripts of approximately 2.3 kb accumulated during senescence of the broccoli florets. The accumulation pattern was similar for both acid invertases but the level of accumulation was greater for the BoINV2 encoded transcript. The accumulation of these transcripts was delayed by water, sucrose and CA treatments compared to the controls. Southern analysis experiments demonstrated that the two cloned acid invertases are part of a small gene family. These findings suggest that acid invertase may be one of the key enzymes responsible for the reduction in sucrose levels that accompanies the rapid deterioration of harvested broccoli.

Characterization of the harvest-induced expression of β-galactosidase in Asparagus officinalis

Abstract The harvest-induced senescence of asparagus spears is accompanied by both up-regulated and down-regulated gene expression. The expression of pTIP31, coding for asparagus β -galactosidase (EC 3.2.1.23) is temporally associated with removal of the asparagus spear from the main body of the plant — neither wounding or compression treatments induce up-regulation of transcripts corresponding to pTIP31. Harvest-induced pTIP31 transcripts appear initially in cells below the meristems of the side branches, and in inner bracts and developing flowers of the spear tip, and in the cells of the outer cortex and conjunctive tissue lower down the spear. Transcripts are also located in vascular tissues 24 h after harvest. Enhanced levels of β -galactosidase activity are found in the branches and bracts of the spear tip within 12 h of harvest, while the rate of increase in activity in the middle zone of the spear was comparable to that of the branches and bracts after 24 h. The proportion of galactose in spear cell walls decreases after harvest. We propose that galactosidase activity releases galactose residues that may be used as respiratory substrate for the rapidly deteriorating asparagus spears.

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.

Xyloglucan endotransglycosylase: a role after growth cessation in harvested asparagus

Little is known about the mode of xyloglucan endotransglycosylase (XET) activity in cell walls once the turgor, which drives expansion, is reduced. Such a situation exists when growing shoots are excised from the parent plant, and is the case for many commercially valuable vegetable crops, e.g. asparagus, Asparagus officinalis L. XET activity was present in all zones of rapidly growing, immature asparagus spears, but with highest levels at the spear base where elongation growth had ceased. Activity increased in all parts of the spear for up to 72 h after harvest. Two members of the XET-related gene family in asparagus (AoXET1 and AoXET2) were isolated and mRNA corresponding to these clones accumulated at low levels, particularly in the basal zone during spear growth. Transcript levels increased in all parts of the asparagus spear after harvest, but this increase did not coincide with the increase in XET activity. The harvest-related changes to xyloglucan molecular weight were restricted to slight, segment-specific, up- or down-shifts. However, this may hide strategic alterations to linkages leading to a more rigid wall without major changes in overall molecular weight. The initial postharvest surge in XET activity could be related to harvest stresses such as water deficit, but we propose that the later induction of AoXET1 and AoXET2 is linked to the development of lignified secondary cell walls.

Inhibition of hexokinase and expression of asparagine synthetase and p‐galactosidase genes during sugar feeding and starvation of asparagus (Asparagus officinalis) callus cultures

Abstract Hexokinase has an important role in hexose metabolism and in signalling sugar status in plants. The aim of this study was to inhibit hexose phosphorylation using a hexokinase inhibitor (glucosamine), and to determine the effects on expression of asparagine synthetase (AS) and (3‐galactosidase during glucose feeding and starvation of asparagus (Asparagus officinalis L.) callus cultures. After 48 h without glucose and a further 24‐h incubation with a range of hexoses and analogues, expression of both AS and β‐galactosidase was repressed by d‐glucose, d‐galactose, and 2‐deoxyglucose, but the genes were not repressed when glucose was absent, or when 3‐O‐methylglucose or L‐glucose were supplied. Glucose‐and fructose‐phosphorylating activity was determined in extracts from callus cultures which had been exposed to glucose, 2‐deoxyglucose, 2‐deoxyglucose with glucosamine or mannitol (as an osmotic control), or glucose‐free media. After 48 h on glucose‐free media and 48‐h incubation with 2‐deoxyglucose and glucosamine, glucose‐ and fructose‐phosphorylating activities were reduced by 68 and 83%, respectively. When glucose was present in the cultures, there was no expression of AS transcripts, but when glucose was absent, AS was highly expressed. AS expression was reduced when 2‐deoxyglucose was present for 48 h, even when glucosamine or mannitol was also present in the culture media. P‐galactosidase was highly expressed when glucose was absent, but expression was very low in all of the treatments which contained 2‐deoxyglucose (including the glucosamine and mannitol treatments). The results suggest AS and P‐galactosidase are sugar regulated, but inconsistencies, particularly reduced AS expression in the presence of glucosamine, are discussed in relation to the possibilities that multiple forms of hexokinases exist which might be differentially affected by glucosamine, and that uptake and distribution of 2‐deoxyglucose and glucosamine are limited.

Protein synthesis inhibitors accelerate the postharvest senescence of asparagus and induce tiprot

Abstract In an attempt to delay the postharvest senescence of asparagus (Asparagus officinalis L.), protein synthesis inhibitors were fed to spears by immersing their butt ends in solutions of either actinomycin D or cycloheximide. These treatments, which are known to retard the senescence of excised flowers and leaves, accelerated the postharvest senescence of spears rather than delaying it. Shelf‐life and spear elongation were both reduced. Spear tips had lower concentrations of hexose and asparagine, and an increased incidence of the postharvest disorder, tiprot, than did tips of control (water‐fed) spears. The significance of these observations on the etiology of tiprot is discussed.