Australian consumer awareness of health benefits associated with vegetable consumption.

AIM: The present study investigated the perceived health benefits of specific vegetable consumption to guide the use of nutrition and health claims on vegetable marketing collateral. METHODS: Free elicitation and consumer ranking data were collected through an online survey of 1000 adults from across Australia and analysed for the perceived importance of vegetables in the daily diet, number of serves consumed per day, knowledge about health-related benefits of specific vegetables and perceived health benefits of vegetable consumption. RESULTS: The importance of vegetables in the diet and daily vegetable consumption was higher in people from an English-speaking background, females, people aged 45 years and over and people living in non-metropolitan areas. Digestion was selected as the major health benefit from consumption of specific vegetables. However, understanding of the health benefits of specific vegetable consumption was relatively low among consumers. Half of the respondents were not sure of the health benefits associated with specific vegetables, except for carrots and spinach. Some respondents volunteered nutrient content or other information. CONCLUSIONS: There was no clear indication that consumers understand the specific health benefits conferred by consumption of vegetables. Nutrient and health benefit labelling therefore has the capacity to enhance knowledge of vegetable consumers. It is recommended that health benefit labelling be tailored to promote greater consumption of vegetables in those demographic groups where vegetable consumption was lower. The present study assists the Australian vegetable industry in helping consumers make more informed consumption choices.

Cell death patterns in Arabidopsis cells subjected to four physiological stressors indicate multiple signalling pathways and cell cycle phase specificity.

Corpse morphology, nuclear DNA fragmentation, expression of senescence-associated genes (SAG) and cysteine protease profiles were investigated to understand cell death patterns in a cell cycle-synchronised Arabidopsis thaliana cell suspension culture treated with four physiological stressors in the late G2 phase. Within 4 h of treatment, polyethylene glycol (PEG, 20 %), mannose (100 mM) and hydrogen peroxide (2 mM) caused DNA fragmentation coinciding with cell permeability to Evans Blue (EB) and produced corpse morphology corresponding to apoptosis-like programmed cell death (AL-PCD) with cytoplasmic retraction from the cell wall. Ethylene (8 mL per 250-mL flask) caused permeability of cells to EB without concomitant nuclear DNA fragmentation and cytoplasmic retraction, suggesting necrotic cell death. Mannose inducing glycolysis block and PEG causing dehydration resulted in relatively similar patterns of upregulation of SAG suggesting similar cell death signalling pathways for these two stress factors, whereas hydrogen peroxide caused unique patterns indicating an alternate pathway for cell death induced by oxidative stress. Ethylene did not cause appreciable changes in SAG expression, confirming necrotic cell death. Expression of AtDAD, BoMT1 and AtSAG2 genes, previously shown to be associated with plant senescence, also changed rapidly during AL-PCD in cultured cells. The profiles of nine distinct cysteine protease-active bands ranging in size from ca. 21.5 to 38.5 kDa found in the control cultures were also altered after treatment with the four stressors, with mannose and PEG again producing similar patterns. Results also suggest that cysteine proteases may have a role in necrotic cell death.

Regreening in spathes of Zantedeschia after anthesis: its physiology and control by fructification and hormones.

The mature pigmented spathe of Zantedeschia is characterized by a developmental process, wherein the spathe regreens after anthesis and prior to senescence of the inflorescence. Previous research has shown that spathe regreening involves redifferentiation of chloroplasts and re-accumulation of chlorophyll, but the detailed physiological changes associated with regreening are still largely unknown. Using Zantedeschia aethiopica and the Zantedeschia pentlandii variety 'Best Gold' as models, this study explores the physiological mechanism and possible roles of fructification, 6-benzylaminopurine (BAP) and gibberellin (GA3 ) in induction or progression of spathe regreening. Application of BAP stimulated regreening in spathe tissue of 'Best Gold' by enhancing accumulation of carotenoid and chlorophyll, and also increasing stacking of grana. In contrast, GA3 retarded formation of double-membrane lamella during chloroplast redifferentiation, thus delaying the onset of regreening. We suggest that these actions of BAP and GA3 have a synergistic effect in delaying the onset of regreening in 'Best Gold' so that when applied together retardation of chlorophyll accumulation, chloroplast redifferentiation and accumulation of carotenoids were enhanced. The elimination of fructification did not prevent the occurrence of regreening in either Zantedeschia model plants, indicating that fructification was not a prerequisite for the induction of regreening. It is still unclear how regreening in Zantedeschia is triggered. We propose that the onset of regreening in Zantedeschia is likely to be a genetically programmed event.

Quantitative Raman spectroscopy for the analysis of carrot bioactives.

Rapid quantitative near-infrared Fourier transform Raman analyses of the key phytonutrients in carrots, polyacetylenes and carotenoids, are reported here for the first time. Solvent extracts of 31 carrot lines were analyzed for these phytonutrients by conventional methods, polyacetylenes by GC-FID and carotenoids by visible spectrophotometry. Carotenoid concentrations were 0-5586 µg g(-1) dry weight (DW). Polyacetylene concentrations were 74-4846 µg g(-1) DW, highest in wild carrots. The polyacetylenes were falcarinol, 6-1237 µg g(-1) DW; falcarindiol, 42-3475 µg g(-1) DW; and falcarindiol 3-acetate, 27-649 µg g(-1) DW. Strong Raman bands for carotenoids gave good correlation to results by visible spectrophotometry. A chemometric model capable of quantitating carotenoids from Raman data was developed. A classification model for rapidly distinguishing carrots with high and low polyacetylene (limit of detection = 1400 µg g(-1)) concentrations based on Raman spectral intensity in the region of 2250 cm(-1) was produced.

Resource partitioning in relation to cohabitation of Lactobacillus species in the mouse forestomach.

Phylogenetic analysis of gut communities of vertebrates is advanced, but the relationships, especially at the trophic level, between commensals that share gut habitats of monogastric animals have not been investigated to any extent. Lactobacillus reuteri strain 100-23 and Lactobacillus johnsonii strain 100-33 cohabit in the forestomach of mice. According to the niche exclusion principle, this should not be possible because both strains can utilise the two main fermentable carbohydrates present in the stomach digesta: glucose and maltose. We show, based on gene transcription analysis, in vitro physiological assays, and in vivo experiments that the two strains can co-exist in the forestomach habitat because 100-23 grows more rapidly using maltose, whereas 100-33 preferentially utilises glucose. Mutation of the maltose phosphorylase gene (malA) of strain 100-23 prevented its growth on maltose-containing culture medium, and resulted in the numerical dominance of 100-33 in the forestomach. The fundamental niche of L. reuteri 100-23 in the mouse forestomach can be defined in terms of 'glucose and maltose trophism'. However, its realised niche when L. johnsonii 100-33 is present is 'maltose trophism'. Hence, nutritional adaptations provide niche differentiation that assists cohabitation by the two strains through resource partitioning in the mouse forestomach. This real life, trophic phenomenon conforms to a mathematical model based on in vitro bacterial doubling times, in vitro transport rates, and concentrations of maltose and glucose in mouse stomach digesta.

Structure and functions of exopolysaccharide produced by gut commensal Lactobacillus reuteri 100-23.

Lactobacillus reuteri strain 100-23 together with a Lactobacillus-free mouse model, provides a system with which the molecular traits underpinning bacterial commensalism in vertebrates can be studied. A polysaccharide was extracted from sucrose-containing liquid cultures of strain 100-23. Chemical analysis showed that this exopolysaccharide was a levan (ß-2, 6-linked fructan). Mutation of the fructosyl transferase (ftf) gene resulted in loss of exopolysaccharide production. The ftf mutant was able to colonise the murine gastrointestinal tract in the absence of competition, but colonisation was impaired in competition with the wild type. Biofilm formation by the mutant on the forestomach epithelial surface was not impaired and the matrix between cells was indistinguishable from that of the wild type in electron micrographs. Colonisation of the mouse gut by the wild-type strain led to increased proportions of regulatory T cells (Foxp3+) in the spleen, whereas colonisation by the ftf mutant did not. Survival of the mutant in sucrose-containing medium was markedly reduced relative to the wild type. Comparison of the genomic ftf loci of strain 100-23 with other L. reuteri strains suggested that the ftf gene was acquired by lateral gene transfer early in the evolution of the species and subsequently diversified at accelerated rates. Levan production by L. reuteri 100-23 may represent a function acquired by the bacterial species for life in moderate to high-sucrose extra-gastrointestinal environments that has subsequently been diverted to novel uses, including immunomodulation, that aid in colonisation of the murine gut.

Controlled atmosphere treatment of broccoli after harvest delays senescence and induces the expression of novel BoCAR genes.

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.

Ethylene-sensitivity regulates proteolytic activity and cysteine protease gene expression in petunia corollas.

To investigate ethylene's role in petal senescence, a comparative analysis of age-related changes in total protein, protease activity, and the expression of nine cysteine protease genes in the corollas of ethylene-sensitive Petuniaxhybrida cv. Mitchell Diploid (MD) and ethylene-insensitive (35S:etr1-1; line 44568) transgenic petunias was conducted. The later stages of corolla senescence in MD flowers were associated with decreased fresh weight, decreased total protein, and increased proteolytic activity. Corolla senescence was delayed by approximately 8 d in etr-44568 transgenic petunias, and decreases in corolla fresh weight, protein content, and maximum proteolytic activity were similarly delayed. Protease inhibitor studies indicated that the majority of the protease activity in senescing petals was due to cysteine proteases. Nine cysteine proteases expressed in petals were subsequently identified. Northern blot analysis indicated that six of the nine cysteine proteases showed increased transcript abundance during petal senescence. One of these cysteine proteases, PhCP10, was detected only in senescing tissues. Expression of four of the senescence-associated cysteine proteases was delayed, but not prevented in etr-44568 flowers. The other two senescence associated cysteine proteases had high levels of transcript accumulation in etr-44568 corollas at 8 d after flower opening, when MD flowers were senescing. These patterns suggest that age-related factors, other than ethylene, were regulating the up-regulation of these genes during flower ageing. The delay in visible symptoms and biochemical and molecular indicators of senescence in ethylene-insensitive flowers is consistent with the concept that ethylene modulates the timing of senescence pathways in petals.

Galactosidases in opening, senescing and water-stressed Sandersonia aurantiaca flowers.

Three glycosyl hydrolase family 35 ß-galactosidase-encoding cDNAs, SaGAL1 (full-length), SaGAL2 and SaGA3L (both partial), have been isolated from Sandersonia aurantiaca (Hook.) SaGAL1 protein was functionally expressed in E. coli and ß-galactosidase identity confirmed by activity assay. All three clones are primarily expressed in tepal tissues of senescing sandersonia flowers. In order to identify relationships between tepal texture and galactose metabolism, cut sandersonia flowers were treated with sucrose, periods of dryness or PEG and parameters associated with galactose metabolism and firmness were monitored. Sucrose supplementation, known to increase tepal firmness, delayed expression of SaGAL1 and SaGAL3 in opening (stage 5) flowers, whereas the response to periods of dryness followed by rehydration depended on the maturity of the flower. These treatments also tended to hasten the onset of processes associated with programmed cell death, monitored by PRT5 (a senescence-associated protease) expression. Galactosidase activity and cell wall galactose content were also affected but in an inconsistent manner. PEG supplied to opening flowers for 1 d followed by water, induced a long period of wilt, and intensive PRT5 expression. However, ß-galactosidase gene expression and activity was delayed in these flowers, and cell-wall galactose content changed apparently independently of galactosidase activity. We have not been able to demonstrate a causal connection between the change in petal texture and concurrent induction of galactose mobilisation in sandersonia during normal development and senescence.

Evaluation of carbohydrates in Pukekohe Longkeeper and Grano cultivars of Allium cepa.

The storage, soluble, and structural carbohydrates of two onion cultivars, the hard, pungent Pukekohe Longkeeper (PLK) and the softer, milder Houston Grano, were analyzed to determine differences that might be related to their response to sulfur nutrition received during growth as well as their postharvest attributes and end-use suitability. PLK tissue contained 1.37 times more dry matter than Grano and was composed of more fructan and sucrose and less glucose and fructose than Grano [corrected] There were also differences in neutral sugar content, especially galactose, and the amount, size, and content of pectin fractions soluble in chelator and weak alkali. These two onion cultivars differed in their capacity to take up sulfur, but there was no statistical association between sulfur supply and any measured dry matter component.

Molecular analysis of programmed cell death during senescence in Arabidopsis thaliana and Brassica oleracea: cloning broccoli LSD1, Bax inhibitor and serine palmitoyltransferase homologues.

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.

Identification of dehydration-responsive cysteine proteases during post-harvest senescence of broccoli florets.

Harvest-induced senescence of broccoli results in tissue wilting and sepal chlorosis. As senescence progresses, chlorophyll and protein levels in floret tissues decline and endo-protease activity (measured with azo-casein) increases. Protease activity increased from 24 h after harvest for tissues held in air at 20 degrees C. Activity was lower in floret tissues from branchlets that had been held in solutions of sucrose (2% w/v) or under high carbon dioxide, low oxygen (10% CO(2), 5% O(2)) conditions. Four protease-active protein bands were identified in senescing floret tissue by zymography, and the use of chemical inhibitors of protease action suggests that some 44% of protease activity in senescing floret tissue 72 h after harvest is due to the action of cysteine and serine proteases. Four putative cysteine protease cDNAs have been isolated from broccoli floret tissue (BoCP1, BoCP2, BoCP3, BoCP4). The cDNAs are most similar (73-89% at the amino acid level) to dehydration-responsive cysteine proteases previously isolated from Arabidopsis thaliana (RD19, RD21). The mRNAs encoded by the broccoli cDNAs are expressed in floret tissue during harvest-induced senescence with mRNA accumulating within 6 h of harvest for BoCP1, 12 h of harvest for BoCP4 and within 24 h of harvest for BoCP2 and BoCP3. Induction of the cDNAs is differentially delayed when broccoli branchlets are held in solutions of water or sucrose. In addition, the expression of BoCP1 and BoCP3 is inhibited in tissue held in atmospheres of high carbon dioxide/low oxygen (10% CO(2), 5% O(2)). The putative cysteine protease mRNAs are expressed before measurable increases in endo-protease activity, loss of protein, chlorophyll or tissue chlorosis.

Programmed cell death during flower senescence: isolation and characterization of cysteine proteinases from Sandersonia aurantiaca.

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 31kDa). 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.