Donald E. Irving

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.

Identification of intracellular calcium oxalate crystals in Chamelaucium uncinatum (Myrtaceae)

Intracellular inclusions in the pedicel and calyx-tube tissues of Chamelaucium uncinatum Schauer (Myrtaceae) flowers are irregular in shape. They were shown, by polarised light and scanning electron microscopy, to be birefringent 8.9–29.5 μm druse (i.e. aggregate) crystals. Energy-dispersive X-ray spectroscopy showed that these crystals were predominantly composed of calcium. Histochemical and acid-solubility tests indicated that the crystals were calcium oxalate. Raman microprobe spectroscopy was used to confirm this chemical identity. The calcium oxalate crystals were located in xylem-vessel lumens and also in parenchyma cells adjacent to vascular tissues. Thus, the crystals may function to regulate soluble calcium concentrations in C. uncinatum tissues near sites where calcium is unloaded from the xylem.

Characterisation of xylem conduits and their possible role in limiting the vase life of cut Acacia holosericea (Mimosaceae) foliage stems

Early desiccation limits the vase life of Acacia cut flowers and foliage and may be attributable to poor hydraulic conductivity (Kh) of the cut stems. Acacia holosericea A.Cunn. ex G.Don has been adopted as the test species to investigate the postharvest water relations of the genus Acacia. To understand potential constraints on Kh, xylem conduits in cut A. holosericea stems were anatomically characterised by light and scanning and transmission electron microscopy. Vessels with simple perforation plates and tracheids were the principal water conducting cells. Bordered vestured intervessel pits were present in xylem vessel elements. The majority of conduits (89%) were short at 1-5cm long. Only 2% were 15-16cm in length. Mean xylem conduit diameter was 77±0.9µm and the diameter profile showed a normal distribution, with 29% of diameters in the range of 70-80µm. Simple perforation plates can offer relatively low resistance to water flow. On the other hand, bordered vestured pits and short xylem conduits can confer comparatively high resistance to water flow. Overall, the presence of bordered vestured pits, together with a high proportion of short xylem conduits and high stomatal densities (232±2mm-2) on unifacial phyllodes, could contribute to early dehydration of A. holosericea cut foliage stems standing in vase water. Further research will relate these anatomical features with changes in Kh and transpiration of cut foliage stems.

Anatomy of ethylene-induced floral-organ abscission in Chamelaucium uncinatum (Myrtaceae)

Postharvest abscission of Geraldton waxflower (Chamelaucium uncinatum Schauer) flower buds and flowers is ethylene-mediated. Exposure of floral organs to exogenous ethylene (1 mu L L-1) for 6 h at 20 degrees C induced separation at a morphologically and anatomically distinct abscission zone between the pedicel and. oral tube. Flower buds with opening petals and flowers with a nectiferous hypanthium were generally more responsive to exogenous ethylene than were flower buds enclosed in shiny bracteoles and aged (senescing) flowers. The anatomy of abscission-zone cells did not change at sequential stages of floral development from immature buds to aged flowers. The zone comprised a layer of small, laterally elongated-to-rounded, closely packed and highly protoplasmic parenchyma cells. Abscission occurred at a two- to four-cell-wide separation layer within the abscission zone. The process involved degradation of the middle lamella between separation layer cells. Following abscission, cells on both the proximal and distal faces of the separation layer became spherical, loosely packed and contained degenerating protoplasm. Central vascular tissues within the surrounding band of separation layer cells became torn and fractured. For flower buds, bracteoles that enclose the immature floral tube also separated at an abscission zone. However, this secondary abscission zone appeared less sensitive to ethylene than the primary ( central). oral-tube abscission zone as bracteoles generally only completely abscised when exposed to 10 mu L L-1 ethylene for the longer period of 24 h at 20 degrees C. The smooth surfaces of abscised separation-layer cells suggest that hydrolase enzymes degrade the middle lamella between adjacent cell walls.

Field applications of three different classes of known host plant defence elicitors did not suppress infection of Geraldton waxflower by Botrytis cinerea

Ethylene-mediated flower abscission caused by Botrytis infection afflicts cut Geraldton waxflower stems. Preharvest spray applications of three known host plant defence elicitors, benzothiadiazole (BTH), methyl jasmonate (MeJA) or silicon (Si), were applied to Geraldton waxflower cvv. Mullering Brook and My Sweet Sixteen. Their individual efficacy in postharvest suppression of Botrytis disease developmentwas assessed. Field applications of BTH or Si generally had no significant (P>0.05) effect on Botrytis disease severity on either cultivar. MeJA sprays did not significantly (P>0.05) reduce disease severity on cv. Mullering Brook, but slightly and significantly (P<0.05) suppressed Botrytis on cv. My Sweet Sixteen at concentrations of 500 and 750 μM MeJA. One Si treatment, 1500 mg SiO2/L, significantly (P<0.05) reduced floral abscission on cv. Mullering Brook. Overall, field applications of these three host plant defence elicitor chemicals as spray treatments had little effect on vase life, water uptake and relative fresh weight of the cut flower sprigs. Moreover, they did not suppress Botrytis or associated postharvest floral abscission in cut Geraldton waxflower flowers.

Expression of asparagine synthetase in response to carbohydrate supply in model callus cultures and shoot tips of asparagus (Asparagus officinalis L.)

Sugar uptake and metabolism were studied in callus cultures and shoot tips of asparagus. Asparagus callus cultures were used to model senescence in shoot tips. Callus cultures absorbed glucose from a nutrient medium, and accumulated sucrose, glucose and fructose. This uptake of glucose by the callus cultures down-regulated expression of asparagine synthetase and beta -galactosidase transcripts that otherwise accumulated when sugar was withheld. When 80 mm-long asparagus shoots were excised from growing plants and placed in 2% and 8% sucrose solutions, endogenous concentrations of sucrose, glucose, fructose, UDPglucose, and glucose-6-phosphate declined in the 30mm-long meristematic tip regions. At the same time, asparagine and asparagine synthetase gene transcripts began to accumulate in these tips. When 10 mm-long asparagus shoot tips were placed on glucose- or fructose-containing agar, the tips accumulated sucrose, glucose and fructose, and asparagine accumulation and expression of asparagine synthetase were marginally reduced. We concluded that in callus cultures, asparagine synthetase expression was sugar regulated, but that sugar regulation was not as pronounced in asparagus shoot tips. This may be due in part to slower rates of sugar uptake into shoot tips and in part to compartmentation of sugars in the tips. We suggest that callus cultures are not a suitable model for metabolic studies in asparagus shoot tips.

Sensitivity of Geraldton waxflower to ethylene-induced flower abscission is reduced at low temperature

Summary Exposure to ethylene gas elicits flower abscission from cut stems of Geraldton waxflower (Chamelaucium uncinatum Schauer). Ethylene response rates in plants are mediated by temperature. At 20°C, flower abscission from waxflower ‘Purple Pride’ occurred upon 12 h exposure to 1 µ11–1 ethylene. This ethylene treatment did not cause flower abscission at either 10 or 2°C. Moreover, flowers held at 2°C were insensitive to 48 h exposure to 1, 10 and 100 µ11–1 ethylene. However, increasing the duration of treatment with 1 µ11–1 ethylene at 10 and 2°C to 48 and 144 h, respectively, induced flower abscission. When flowers were held at 20°C in air without exogenous ethylene following continuous exposure to 1 µ11–1 ethylene at 2°C, the duration required to elicit flower abscission was reduced from 144 to 72 h. Collectively, these responses show that maintaining harvested waxflower at low temperature (e.g. 2°C) is an effective means to minimise ethylene-mediated flower abscission.

Effects of multiple applications of chemical elicitors on Botrytis cinerea infecting Geraldton waxflower

Geraldton waxflower is susceptible to postharvest floral abscission caused by Botrytis cinerea infection. Pot- and field-grown waxflower cv. Mullering Brook plants and/or their harvested sprigs were spray treated with three known host plant defence elicitors, methyl jasmonate (MeJA), benzothiadiazole (BTH) or silicon (Si), and challenged with Botrytis cinerea after harvest. The efficacy of multiple MeJA, MeJA combined with BTH or Si and combined preand postharvest MeJA treatments in suppressing B. cinerea disease and floral abscission was assessed. Preharvest foliar applications of MeJA (1000 μM; two or four times), MeJA combined with BTH (150 mg/L) and MeJA combined with Si (1500 mg SiO2/L) generally did not suppress B. cinerea development and floral abscission from harvested sprigs. MeJA plus Si applied to pot-grown plants slightly and significantly (P<0.05) increased disease incidence and severity and flower fall. However, application of a MeJA postharvest (1000 μM) spray or pre- (1000 μM) plus postharvest (500 μM) MeJA sprays, significantly (P<0.05) reduced B. cinerea incidence and severity on sprigs from field-grown plants. Overall, the preharvest plus postharvest 1000 μMMeJA spray treatment consistently and significantly (P<0.05) suppressed B. cinerea infection on flowers from both pot- and field-grown plants. However, there was little or no impact of these treatments on floral abscission. Thus, multiple applications of chemical elicitors do not show promise as an approach to controlling B. cinerea-induced postharvest floral abscission in Geraldton waxflower.

Post-harvest browning syndrome and other qualities and defects in Backhousia myrtifolia

Summary Backhousia myrtifolia is a species native to Australia that shows potential as a cut flower crop. During Spring and Summer, it bears numerous small florets with prominent white sepals and glossy deep-green foliage. B. myrtifolia is harvested either when tight white buds are present in the centre of the star-shaped sepals, or following bud burst, after the petals and stamens have abscised to leave only the sepals.Wilting and brown-to-black discolouration of the flowers and foliage can markedly reduce stem quality. Several forms of discolouration were characterised over the 2004 – 2006 flowering seasons and were collectively termed ‘post-harvest browning syndrome’. Further research based on the symptomatology described herein is required to elucidate the causal agent(s).

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.