Tip Of Spear Research Articles

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.

Tiprot in asparagus: effect of temperature during spear growth

Abstract Tiprot, a postharvest disorder of asparagus, occurred at high frequency (99%) in spears grown at 20 °C in controlled temperature conditions, but at low frequency (3%) in spears grown at 13 °C. During spear growth, if temperatures of spears and crowns were controlled separately at 13 and 20 °C or at 20 and 13 °C, respectively, incidence of tiprot was similar at 56–62%. The occurrence of tiprot was negatively correlated with sucrose content of the spear tip, and positively correlated with spear tip respiration rate and spear height growth rate. Sucrose content of the spear tip at harvest gives the most practical measure of predisposition to tiprot during storage but may not be the primary cause of the disorder.

Asparagine synthesis and localization of transcripts for asparagine synthetase in tips of harvested asparagus spears

Summary Asparagine (Asn) accumulated markedly in the tips of harvested asparagus (Asparagus officinalis L.; King et al., 1990) shortly after the harvest-induced accumulation of asparagine synthetase (AS) transcripts (Davies and King, 1993). Supplying harvested asparagus spears with 1 mmol-L−1 phosphinothricin (PPT, a specific inhibitor of glutamine synthetase) markedly reduced glutamine (Gln) content, increased the ammonium content, and prevented Asn formation in spear tips from 6 to 12 h after harvest. AS transcripts accumulated to high levels in spears supplied with either PPT or water from 6 h after harvest. The data provide evidence that the in vivo activity of AS is strictly Gin-dependent. In situ localization of AS transcripts demonstrated tissue-specific responses by cells of the spear tip to harvest. We propose that Asn accumulation effectively acts as a nitrogen storage mechanism in the tips of harvested asparagus spears.

Cloning of a harvest-induced beta-galactosidase from tips of harvested asparagus spears.

We are investigating the early changes in physiology, biochemistry, and gene expression that follow harvest of asparagus (Asparagus officinalis L.) spears to identify factors regulating the postharvest senescence of horticultural crops harvested while physiologically immature. The tips of spears comprise immature developing tissues that appear particularly susceptible to harvest stress, and they are usually the first parts of the spear to show symptoms of postharvest senescence, including feathering and browning of bracts, tissue flaccidity, and cellular breakdown (King et al., 1990). These visual symptoms of senescence are preceded by rapid changes in carbon and nitrogen metabolism and gene expression. Within 48 h of harvest, the respiration rate of tips declines markedly, protein is lost, and both free amino acids (especially Asn) and ammonia accumulate (King et al., 1990). Major changes in gene expression occur within 6 h of harvest, including the de novo induction of specific genes (King and Davies, 1992). We previously constructed cDNA libraries from mRNA extracted from tips of spears at harvest and from tips of spears held in the dark at 20°C for 12 h. Differential hybridization screening of these libraries isolated nine cDNA clones whose transcripts had altered expression in tips of harvested asparagus spears (King and Davies, 1992), including a cDNA clone for a harvest-induced Asn synthetase (King and Davies, 1992; Davies and King, 1993). We report here the cloning and characteristics of a full-length cDNA (pTIP31) encoding P-galactosidase using one of our original harvest-induced partial cDNA clones (pTIPl1) as a hybridization probe (Table I). The derived protein sequence of pTIP31 shares 71% amino acid identity with ABGl, a cDNA clone encoding a member of the apple P-galactosidase gene family (Ross et al., 1994), and 63% amino acid identity with DINCARSR12, an ethylene-regulated P-galactosidase cDNA clone isolated from a senescing carnation peta1 library (Ragothama et al., 1991; W.R. Woodson, personal communication). Transcripts homologous to pTIP31 increase in abundance in tips of harvested asparagus spears. Induction of P-galactosidase may explain the observation of Waldron and Selvendran (1990), who noted a 50% reduction in the levels of

Mineral Nutrient Composition of Green and White Asparagus Spears

Mineral nutrients were determined in green and white `Jersey Giant' asparagus (Asparagus officinalis L.) spears grown in 1991 near Booneville, Ark. Green spears had higher concentrations of tissue N, K, P, S, Na, and Zn; similar levels of Fe, Al, and Cu; and lower levels of NO3 than white spears grown under opaque plastic rowcovers or under sawdust mulch. Levels of Ca, Mg, and Mn were higher in plastic-grown white spears than in those grown in sawdust. The spear tip contained higher concentrations of K, S, and Cu than the butt. Less NO3, Fe, and Al were in the tip than in the butt. Spear distribution gradients for K, Fe, and Cu were linear, whereas S and NO3 gradients were both linear and quadratic. Nitrogen, P, Ca, Mg, Zn, Al, and Mn levels were influenced by spear production method and spear segment.

Isoforms of glutamine synthetase in asparagus spears: the cytosolic enzyme increases after harvest

ABSTRACTTwo distinct forms of glutamine synthetase (GS) have been identified in the spear tip tissues of harvested asparagus (Asparagus officinalis L. cv. Limbras 10). The GS activities were separated by anion exchange chromatography. They have distinct kinetic properties and contain polypeptides of different sizes, and the abundances of the GS isoforms change differently after harvest. Plastid GS has a 44 kD polypeptide, and during the post‐harvest period the abundance of this polypeptide declined dramatically. After 5 d, the activity of plastid GS had declined to just 20% of that at harvest. Cytosolic GS has a 40 kD polypeptide and is the major constituent of the GS activity present at harvest (73% of total). After harvest, cytosolic GS activity declined by half and then, at 3 or 4 d after harvest, rose to 80% of the cytosolic GS activity present at harvest. The nitrogen metabolism of asparagus spears is significantly altered as the tissues deteriorate rapidly after harvest. We demonstrate that cytosolic GS activity increases during the post‐harvest period and is likely to be a critical feature of the physiology of the tip of a harvested asparagus spear.

Postharvest lipid loss, malate accumulation, and appearance of malate synthase activity in asparagus spear tips

Lipid levels, glyoxylate-cycle enzymes and respiratory quotients were measured in 3 cm tips of asparagus spears (Asparagus officinalis L.) during 5 days of storage at 20°C, to determine whether lipids are mobilised as in some other senescing plant tissues. Within 12 h, the respiratory quotient dropped from near unity to 0.66 before stabilising around 0.75 after 2 days. Over 5 days of storage, almost half of the total lipid in spear tips was lost. Malate synthase (EC 4.1.3.2) activity, which was not detectable at harvest, appeared after 1–2 days and continued to increase throughout the remaining 3 days. Malate concentration increased 3- to 4-fold in 4–5 days. Isocitrate lyase (EC 4.1.3.1) activity was not detected at any time during storage, and it is unlikely that the glyoxylate cycle functions in tips of harvested asparagus spears. These results are discussed in relation to other postharvest metabolic events in asparagus.

Asparagus: Variation amongst plants in susceptibility to the postharvest disorder, tip breakdown

Abstract Tip breakdown is a serious postharvest disorder of asparagus (Asparagus offtcinalis) which can result in severe wastage and quality loss. We report significant variation amongst plants in their susceptibility to this disorder. This variation may be genetic in origin, but the results presented are equivocal on this. Plants with low levels of tip breakdown had higher levels of soluble carbohydrate in spear tips than plants susceptible to the disorder. Protein levels and postharvest accumulation of ammonia were not significantly different in plants with low levels of tip breakdown compared to plants with high levels. The possibilities for selecting resistant plants are discussed.

Sucrose synthase, invertases, and sugars in growing asparagus spears

Abstract Asparagus (Asparagus officinalisL.) spear tips lose sucrose rapidly after harvest and this probably contributes to spears’ short postharvest life. As the first step towards controlling this loss of sucrose and possibly improving postharvest life, the activities of sucrose‐cleaving enzymes, and levels of sugars were measured in tips and 3 cm sections of spears from tip to butt. Sucrose was the major sugar in tips but not in the rest of the spear. Glucose and fructose were almost equimolar throughout the spear and increased fivefold from tip to butt. Fructose was always slightly more abundant than glucose. Sucrose synthase, bound acid invertase, and neutral invertase activities were similar in both tips and spear sections. Soluble acid invertase activity in the middle section of the spear was 4—5 times that of tips and butts. Sugar composition and enzyme activities in spear sections and tips were not closely correlated.

Effect of harvest date on the shelf life of asparagus

Abstract Commercial asparagus production occurs over an extended period in spring from carbohydrate reserves built up during the previous summer and stored in the roots. The effect of harvest duration on spear yield and quality, and on the depletion of storage root carbohydrates has been widely studied. However, little work has been reported on the effect of harvest period on the storage life and biochemistry of asparagus spears. Therefore, asparagus spears (Asparagus officinalis L. cv. Limbras 10) were harvested over the commercial harvesting season in 2 years and held at 20°C to assess shelf life and to monitor postharvest biochemistry. Shelf life declined almost linearly in both years from c. 6 days for early‐season spears to c. 3 days for late‐season spears. On the evidence of rates of protein and carbohydrate loss, and ammonia accumulation in the spear tips, the decline in shelf life was not associated with accelerated biochemical changes. Spear fresh weight at harvest (and therefore spear thickness)...

Spatial distribution of abscisic acid and 2-trans-abscisic acid in spears, buds, rhizomes and roots of asparagus ( Asparagus officinalis L.)

Changes in the levels and spatial distribution of abscisic acid (ABA) and 2-trans-abscisic acid (t-ABA) were determined in different sections of spears, buds, rhizomes and roots of dark-grown asparagus ( Asparagus officinalis L. cultivar ‘Mary Washington 500 W’). In both buds and rhizomes, ABA level was higher in the younger regions where buds would sprout, while in the growing spears, there was a declining gradient from the morphological apex towards the base. Spear tips were characterized by constant high levels of ABA during their elongation. The growth rate was low in the tip region, and showed a large gradient, declining from the zone of elongation (located 15–35 mm from the tip) to the butt region. Once lateral buds began their growth, the content of ABA in lateral buds plus scale leaves associated with the tip region, was the highest among all tested tissues. The levels of the physiologically less active t-ABA, on a fresh weight basis, showed a gradient, declining from the tip to the butt region in all parts, but was less in absolute amounts. These results support the hypothesis that ABA is involved in mobilizing carbohydrate reserves for spear growth.

Respiration, soluble carbohydrates and enzymes of carbohydrate metabolism in tips of harvested asparagus spears

We held asparagus ( Asparagus officinalis L.) spears at 20°C for up to 5 days after harvest and examined changes in respiration rate, soluble carbohydrates and the activities of enzymes concerned with glycolysis and carbohydrate breakdwon in the tips. Respiration rate declined after harvest, to reach 25% of its initial rate after 2 days. The gas exchange (respiratory) quotient was 1.05 at harvest and declined in parallel with respiration rate for 2 days. There was a rapid loss of sucrose from spear tips over the first 6 h, and a more gradual loss after that. Glucose concentration declined gradually throughout, but fructose concentration barely changed over 3 days. We conclude that a major change in the metabolism of asparagus spear tips occurs soon after harvest. The change is characterised by a switch from sucrose-derived hexose phosphate to another primary respiratory substrate, probably derived from protein and/or lipid. The activities of hexokinase (EC 2.7.1.1), fructokinase (EC 2.7.1.4), phosphofructikinase (EC 2.7.1.11), sucrose synthase (EC 2.4.1.13) and alkaline invertase (EC 3.2.1.26) were generally low compared with the activities of the other enzymes assayed. Overall, enzyme activities declined over 5 days of starvation. The exceptions were hexokinase, phosphofructokinase and pyruvate kinase (EC 2.7.1.40), where activities were reasonably steady.

Nitrogen metabolism in harvested asparagus: No difference between light and dark storage at 20°C

To extend our knowledge of nitrogen metabolism in asparagus after harvest we monitored protein, amino acid and ammonia levels and glutamine synthetase activity in tip sections (0–30 mm) of asparagus spears ( Asparagus officinalis L. cv. Limbras 10) stored for up to 48 h in continuous light or dark at 20°C. The patterns of change in light and dark were almost identical. Total protein content increased by 20% 12 h after harvest, before declining to harvest levels by 48 h. Ammonia increased slightly by 48 h. Glutamine synthetase activity increased 10–15% by 18 h then declined to 90% of harvest activity by 48 h. Glutamic acid and glutamine were the most abundant amino acids at harvest, accounting for 42% of the pool. Glutamine and proline levels fell rapidly. By 48 h most amino acids had increased substantially, with asparagine accounting for 28% of the pool. Glutamine levels fell steadily during storage, irrespective of the regime. Our results suggest that postharvest nitrogen metabolism in asparagus spear tips is not modulated by light.

Postharvest changes in ammonium, amino acids and enzymes of amino acid metabolism in asparagus spear tips

AbstractHarvested asparagus (Asparagus officinalis L) spears accumulate ammonium and amino acids in their tips during storage at ambient temperatures. To further investigate these phenomena the authors held spears at 20°C in the dark after harvest and examined several parameters of amino acid metabolism in the tips. Soluble protein content declined faster than total protein over the 5‐day storage period. Protein loss was accompanied by an increase in total free amino acids with asparagine showing the most dramatic increase. A pronounced accumulation of ammonium occurred, starting after 3 days. The activities of glutamine synthetase (EC 6.3.1.2) and aspartate and alanine aminotransferases (EC 2.6.1.1 and EC 2.6.1.2, respectively) declined slightly whereas glutamate dehydrogenase (EC 1.4.1.2) activity almost doubled over 5 days. Phenylalanine ammonia lyase (EC 4.3.1.5) activity declined rapidly during the first 2 days and then partially recovered. Asparagine synthetase (EC 6.3.1.1), asparagine aminotransferase (EC 2.6.1.14), asparaginase (EC 3.5.1.1) and glutaminase (EC 3.5.1.2) activities were not detectable at any time during the storage period. Ammonium accumulation could be caused by increased glutamate dehydrogenase activity coupled with a shortage of aspartate.

Identification, cDNA Cloning, and Analysis of mRNAs Having Altered Expression in Tips of Harvested Asparagus Spears

Changes in mRNA activity in tips of harvested asparagus spears (Asparagus officinalis L.) held in light or dark for up to 48 h at 20 degrees C were investigated as an initial step in elucidating the genetic response of asparagus spears to harvest. Total RNA was isolated from 30-mm tips of spears 180 mm in length at 0, 6, 12, 24, and 48 h after spear harvest and translated in vitro, and translation products were separated using both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis. We detected 25 consistent changes in translatable mRNAs, involving both increase and decrease in mRNA abundance. The majority of the changes occurred within 12 h of harvest. Most of the changes were not light regulated. cDNA libraries were constructed from polyadenylated mRNA extracted from tips of spears at harvest (0 h) and after 12 h in the dark at 20 degrees C. Differential hybridization screening of the cDNA libraries isolated nine cDNA clones whose corresponding transcripts had altered expression after harvest. Investigations of mRNA activity during spear development demonstrated that the changes detected were harvest related. Possible roles for the mRNAs corresponding to the isolated clones in tips of harvested spears are discussed.

Physiological changes in asparagus spears immediately after harvest

Respiratory activity of asparagus spears measured in the field showed a 4-fold increase between the butt and the tip of 190-mm spears. After harvest, there was an immediate rise in the respiration rate, followed by a rapid drop during 24 h at 16°C to a constant level, ∼ 30% of the peak respiration rate. Strong gradients of sugars and proteins were measured along the spears with low levels of sugars and high levels of proteins present in the spear tips. Sugars declined markedly in the first 24 h after harvest, particularly in spear tips. Proteins in spear tips were unchanged 24 h after harvest, but had decreased 25% by 72 h. Total free amino acids remained steady for the first 24 h after harvest, but increased by 75% at 48 h. Asparagine/aspartic acid increased, whereas glutamine/glutamic acid and proline decreased in concentration substantially during the first 24 h after harvest. Tips of taller spears had a lower sugar content and more protein than tips of short spears.

Physiological changes in asparagus spear tips after harvest

To extend our understanding of the physiology of asparagus after harvest, changes in respiration rate, protein and amino acid complement, and ultrastructure of tip sections (0–30 mm) of asparagus spears (Asparagus officinalis L. cv. Limbras 10) were investigated. Spears had been stored for up to 4 days in the dark at 20°C. Respiration rate (carbon dioxide efflux) declined rapidly after harvest before stabilizing at 12 h at ca 50% of the rate at harvest. Protein, amino acid, and ammonium content of tip sections of 180 mm spears (intact tip sections) during storage, and comparable sections; excised from spears at harvest and subsequently stored (excised tip sections), were compared. Total protein content of intact and excised tip sections increased ca 10% 6–12 h after harvest, and then declined to ca 85% of harvest levels at 48 h. Gel electrophoresis in the presence of sodium dodecyl sulphate revealed the net loss of specific proteins at 48 h. Free amino acid content of excised tip sections declined to ca 75% of harvest levels 12 h after harvest, and then increased to 150% of harvest levels by 48 h. Glutamine levels declined rapidly after harvest, and asparagine content increased ca 200% at 24 h. Similar trends in free amino acid content were found in sections of intact tips. Ammonia (ammonium ions) accumulated to ca 0.3% dry weight at 48 h in both intact and excised tip sections. Ultrastructural studies revealed that tonoplast breakdown commenced 48–96 h after harvest. Results are discussed in relation to the sequence of physiological events following harvest and the timing of mechanisms responsible for their initiation.

Physiological changes in asparagus spear tips after harvest

Physiological changes in asparagus spear tips after harvest

Flavour and metabolic changes in asparagus during storage

Flavour acceptability of apical spear segments declined within 2 days of storage at 1°C and further declined after 7 days of storage. Decline in flavour acceptance was accompanied by off-flavour development, a decrease in soluble carbohydrates and an increase in protein content of spear tip segments. Respiration rate of spears stored at 1 or 20°C declined after harvest before reaching an equilibrium. A significant loss of soluble carbohydrates and proteins from tip segments occurred during storage at 20°C. Storage at 1°C reduced the rate of substrate loss.

Endogenous Gibberellins and Cytokinins in Spear Tips of Asparagus officinalis in Relation to Sex Expression

Abstract Endogenous gibberellins (GA) and cytokinins (CK) were extracted from asparagus (Asparagus officinalis L.) spear tips, purified, and determined by lettuce hypocotyl and amaranthus bioassays, respectively. There was no quantitative difference in GA-Iike activity between heterogametic male and female spears. The major GA fraction in asparagus spears has 1 OH group. Asparagus spears contain 3 major fractions of CK-like activity. Fraction 1 eluted from Sephadex LH-20 and C18 HPLC columns with or before zeatin-riboside. Fractions 2 and 3 eluted in a similar pattern to IPA-riboside and IPA, respectively. There were higher levels of CK fraction 2 and trends toward higher levels of fraction 1 and total CK in female than in heterogametic male spears. There were also higher CK:GA ratios in female than in heterogametic male spears. The data support the hypothesis that sex in asparagus is controlled in part by CK levels or by CK:GA ratios.