Wilhelmina M. Borst

Sugar Regulation of Harvest-Related Genes in Asparagus

The signals controlling the abundance of transcripts up-regulated (pTIP27, pTIP31, and pTIP32) or down-regulated (pTIP20 and pTIP21) after harvest in asparagus (Asparagus officinalis L.) spears were examined. pTIP27 and pTIP31 are known to encode asparagine synthetase (AS) and a [beta]-galactosidase ([beta]-gal) homolog, respectively. The nucleotide sequences of pTIP20, pTIP21, and pTIP32 were determined, and they encode histone 3, histone 2B, and an unknown product, respectively. Changes in respiration, soluble sugars, and abundance of the five mRNAs were similar in the tips stored as 30-mm lengths or as part of 180-mm spears. We previously hypothesized that sugars may regulate the level of AS transcripts in asparagus tissue. Asparagus cell cultures were used to test the role of sugar status in regulating gene expression. Transcript abundance for AS, [beta]-gal, and pTIP32 was low in cells in sugar-containing medium but increased within 12 h after transferring cells to a sugar-free medium. Histone 3 and histone 2B transcripts were, in general, abundant in cells on sugar-containing medium but declined in abundance when transferred to sugar-free medium. When cells were returned to sugar-containing medium the abundance of transcripts for histone 3 and histone 2B increased, whereas that for AS, [beta]-gal, and pTIP32 decreased. Soluble sugar levels are known to decline rapidly in the tips of harvested spears. Metabolic regulation by sugar status may have a major influence on gene expression in asparagus spears and other tissues after harvest.

Similarities in Gene Expression during the Postharvest-Induced Senescence of Spears and Natural Foliar Senescence of Asparagus

Changes in gene expression and tissue composition were investigated during foliar development and natural senescence of asparagus (Asparagus officinalis L.). Three phases in development and senescence of the foliage were characterized: early fern growth, mature fern, and senescence, when a marked loss of chlorophyll, sucrose, and protein occurred and major changes in translatable mRNAs were detected. Transcripts for three asparagus spear harvest-induced cDNA clones, pTIP9, pTIP11, and pTIP12 (G.A. King and K.M. Davies [1992] Plant Physiol 100: 1661–1669), accumulated during natural foliar senescence, suggesting that the underlying regulatory mechanisms may be similar in both developmental situations. We have used our knowledge of asparagus spear physiology, the probable proteins encoded by the cDNA clones, and our fern development data to propose that sugar depletion regulates the accumulation of at least pTIP12 transcripts in senescing asparagus tissue.

Postharvest inhibition of glutamine synthetase activity with phosphinothricin reduces the shelf-life of asparagus

Abstract Harvested spears of asparagus ( Asparagus officinalis L.) accumulate ammonia in their tips towards the end of shelf-life. However, the accumulation of ammonia is not due to limitations in the activity of the ammonia assimilating enzyme, glutamine synthetase (GS). When GS was inhibited by postharvest treatment with phosphinothricin (PPT), the ammonia content of the tips increased almost immediately and the shelf-life of the spears was severely reduced with symptoms of deterioration identical to those commonly ascribed to chilling injury. PPT treatment also inhibited the pattern of glutamine and asparagine accumulation seen during shelf-life. These observations emphasize that during the postharvest life of asparagus GS has a pivotal role in reassimilating considerable amounts (3% of dry weight) of ammonia in the tips.

Glutamine Synthetase Activity, Ammonium Accumulation and Growth of Callus Cultures of Asparagus officinalis L. Exposed to High Ammonium or Phosphinothricin

Summary Glutamine synthetase (GS) activity, ammonium accumulation and growth responses of callus cultures of Asparagus officinalis L. were investigated following 4 weeks exposure to media with added ammonium, and again after a further 4 weeks on a modified basal medium (MBM) containing no added ammonium. Calli grown on MBM supplemented with 40 or 160 mM ammonium for 4 weeks had reduced GS activity, greatly enhanced ammonium content and reduced growth compared with calli exposed to 10 mM added ammonium. When calli were transferred back to MBM, GS activity increased, ammonium content decreased, and growth was enhanced. Phosphinothricin (PPT) was used to endogenously alter the ammonium content of callus tissue. Exposing calli to 10 or 100 μM PPT for 4 weeks reduced GS activity, enhanced ammonium accumulation and reduced growth compared with calli not exposed to PPT, and markedly enhanced callus glutamine content. Calli exposed to 100 pM PPT did not regrow when transferred back to basal media (BM) without added PPT for an additional 4 weeks. We also separated the effect of PPT-induced ammonium accumulation from alterations in tissue amino acid concentrations by adding up to 25 mM glutamine in addition to PPT during the initial 4-week treatment period. Glutamine supplementation overcame the PPT-induced reduction in growth even though GS activity was severely reduced and ammonium accumulated to high concentrations in calli exposed to 100 μM PPT. All calli continued to grow vigorously when transferred back to BM for an additional 4 weeks. The results demonstrate that ammonium accumulation per se was not lethal to asparagus callus tissue, and suggest other effects of using PPT as a selective inhibitor of GS.

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

Abstract 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 life of asparagus (Asparagus officinalis) under warm conditions can be extended by controlled atmosphere or water feeding

Abstract Green asparagus (Asparagus officinalis) is known to respond to controlled atmosphere storage (CA) at 20°C with increased shelf‐life, reduced respiratory rate, slower loss of soluble carbohydrates and protein, and slower accumulation of asparagine. This work investigated the likelihood of interactive effects of storage of spears in CA along with spear feeding solutions, including 2% sucrose. Standing the spears in a feeding solution extended shelf‐life in air but conferred little additional benefit in CA. Feeding the spears with 2% aqueous sucrose reduced asparagine accumulation and protein loss, but gave no visual benefit over water alone. Spears in the feeding solutions gained weight, particularly during the first 2 days after harvest, but weight gain was slower in CA than in air. Sensory assessment indicated that spears held in CA for 6 days had similar flavour and acceptability to spears held in air for 1 day. Spear quality was more strongly influenced by CA than by feeding solutions. Both approaches could assist in asparagus quality retention where a good refrigerated cool chain is not available, but these technologies are technically challenging to apply to air‐freighted asparagus.

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.

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.

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)...

Spear height at harvest influences postharvest quality of asparagus (Asparagus officinalis)

Abstract Asparagus (Asparagus officinalis L.) spears harvested at intervals during a 24‐h cycle exhibited a clear diurnal pattern in postharvest shelf‐life, with spears harvested at 0200 h lasting 1.1 days longer at 20°C than spears harvested at 1400 h. Susceptibility to tip rot did not appear to fluctuate with harvest time during the day, but was clearly influenced by time of season. Early season harvests were less susceptible to tip rot, and short spears (50 mm) were less susceptible than tall spears (250 mm). Tall spears had lower levels of soluble carbohydrate, and much greater proportions of axillary bud tissue than short spears. First signs of tissue damage during tip rot development were observed in floral buds with collapse of the generative tissue. As damage increased, bracts, then vegetative buds, and the main stem axis were affected. Microbial growth developed on the surface of damaged tissue. Starch grains present in the stem axis or pith tissue disappeared within a day of harvest, but starch localised in a distinct band eight cells in from the cuticle remained until Day 5 after harvest. The postharvest management of longer grades of asparagus spears will be more difficult than shorter grades, and spears harvested in cool conditions will be higher quality than spears harvested in warm conditions.