Changes in the content and distribution, in different organs, of the flavour precursors, the S-alk(en)yl-L-cysteine sulphoxides, during seedling development of onions (Allium cepa) grown under light and dark regimes

Flavour precursors accumulated rapidly in onion seedlings ( Allium cepa L. cv. Pukekohe Longkeeper, May and Ryan) after emergence of the cotyledon. (±)-S-1-Propyl-L-cysteine sulphoxide, found mainly in the shoots, was the predominant flavour precursor. (±) Trans -S-1-propenyl-L-cysteine sulphoxide was important during very early seedling growth when very high concentrations were reported in the root and hypocotyl. (±)-S-1-Methyl-L-cysteine sulphoxide, a minor component in seedlings grown under a normal light regime, was significant during early cotyledon development in seedlings grown in the dark. It is concluded that onion seedlings would be a suitable tissue in which to investigate the biosynthetic pathways of the S-alk(en)yl-L-cysteine sulphoxides.     

The biosynthetic pathway of the S-alk(en)yl-L-cysteine sulphoxides (flavour precursors) in species of Allium

Pulse labelling experiments with ³⁵SO₄ ^2- fed for 24h to intact plants (shooted onion sets)of Allium cepa (onion) showed that >70% of the label appeared in the S-alkenyl-L-cysteine sulphoxides within 18h, reached a maximum at 48h and thereafter decreased. The amount of label detected in the -glutamyl peptide fractions was below 20% of the total label at any time. It is concluded that in intact plants (at the growth stage used) the -glutamyl peptides are not the immediate precursors of the S-alkenyl-L-cysteine sulphoxides. The major S-alkenyl-L-cysteine sulphoxide in onion was found to be compartmentalized mainly within the endoplasmatic reticulum.Abbreviations AllCysSO (+)-S-2-propenyl-L-cysteine sulphoxide - MeCysSO (+)-S-methyl-L-cysteine sulphoxide - PrenCysSO trans-(+)-S-1-propenyl-L-cysteine sulphoxide - ProCysSO (+)-S-propyl-L-cysteine sulphoxide     

Qualitative and Quantitative Analysis of Endogenous Jasmonic Acid in Bulbing and Non-Bulbing Onion Plants

Bulb development in onion plants (Allium cepa L.) is consideredto be regulated by bulbing and anti-bulbing hormones. Sincebulbing involves the disruption of microtubules, both jasmonicacid (JA) and methyl jasmonate (JAMe) are candidates for thebulbing hormone because of their microtubule-disrupting activitiesand wide distribution in higher plants. To survey JA and JAMein onion plants, we developed a radioimmunoassay (RIA) for JAMethat is sensitive enough to detect femtomole amounts of JAMe.Using this RIA, we detected JA in leaf blades, leaf sheathsand roots of onion plants, but no JAMe was detected in any tissue.The endogenous levels of JA in leaf blades, leaf sheaths androots of 4-week-old bulbing and non-bulbing onion plants weredetermined by gas chromatography/selected ion monitoring with[²H₂]JA as an internal standard. The amount of JA per plantin leaf sheaths of bulbing onion plants was about three timeshigher than that of non-bulbing onion plants, although the differencein levels of JA in leaf blades between bulbing and non-bulbingonion plants was quite small, and the level of JA in roots ofbulbing onion plants was lower than that of non-bulbing onionplants. However, the relationship between endogenous JA andthe development of onion bulbs remains to be clarified. (Received June 3, 1992; Accepted October 1, 1992)     

Qualitative and Quantitative Analysis of Endogenous Gibberellins in Onion Plants and Their Effects on Bulb Development

Evidence has been reported that bulb development in onion plants (Allium cepa L.) is controlled by endogenous bulbing and anti-bulbing hormones, and that gibberellin (GA) is a candidate for anti-bulbing hormone (ABH). In this study, we identified a series of C-13-H GAs (GA₁₂, GA₁₅, GA₂₄, GA₉, GA₄, GA₃₄, and 3-epi-GA₄) and a series of C-13-OH GAs (GA₄₄, GA₂₀, GA₁ and GA₈) from the leaf sheaths including the lower part of leaf blades of onion plants (cv. Senshu-Chuko). These results suggested that two independent GA biosynthetic pathways, the early-non-hydroxylation pathway to GA₄ (active GA) and early-13-hydroxylation pathway to GA₁ (active GA), exist in onion plants. It was also suggested that GA₄ and GA₁ have almost the same ability to inhibit bulb development in onion plants induced by treatment with an inhibitor of GA biosynthesis, uniconazole-P. The endogenous levels of GA₁ and GA₄, and their direct precursors, GA₂₀ and GA₉, in leaf blades, leaf sheaths, and roots of 4-week-old bulbing and non-bulbing onion plants were measured by gas chromatography/selected ion monitoring with the corresponding [²H]labeled GAs as internal standards. In most cases, the GA levels in long-day (LD)-grown bulbing onion plants were higher than those of short-day (SD)-grown non-bulbing onion plants, but the GA₁ level in leaf blades of SD-grown onion plants was rather higher than that of LD-grown onion plants. Relationship between the endogenous GAs and bulb development in onion plants is discussed.     

Biosynthesis of the flavour precursors of onion and garlic

Onion (Allium cepa), garlic (A. sativum) and other Alliums are important because of the culinary value of their flavours and odours. These are characteristic of each species and are created by chemical transformation of a series of volatile sulphur compounds generated by cleavage of relatively stable, odourless, S-alk(en)yl cysteine sulphoxide flavour precursors by the enzymes alliinase and lachrymatory-factor synthase. These secondary metabolites are S-methyl cysteine sulphoxide (MCSO, methiin; present in most Alliums, some Brassicaceae), S-allyl cysteine sulphoxide (ACSO, alliin; characteristic of garlic), S-trans-prop-1-enyl cysteine sulphoxide (PECSO, isoalliin; characteristic of onion), and S-propyl cysteine sulphoxide (PCSO, propiin; in onion and related species). Information from studies of the transformation of putative biosynthetic intermediates, radiolabelling, and from measurements of sulphur compounds within onion and garlic have provided information to suggest a biosynthetic pathway. This may involve alk(en)ylation of the cysteine in glutathione, followed by cleavage and oxidation to form the alk(en)yl cysteine sulphoxide flavour precursors. There is also evidence that synthesis of the flavour precursors may involve (thio)alk(en)ylation of cysteine or a precursor such as O-acetyl serine. Both routes may occur depending on the physiological state of the tissue. There are indications from the effects of environmental factors, such as the availability of sulphur, that control of the biosynthesis of each flavour precursor may be different. Cysteine and glutathione metabolism are discussed to indicate parallels with Allium flavour precursor biosynthesis. Finally, possible avenues for exploration to determine the origin in planta of the alk(en)yl groups are suggested.     

Studies in the Physiology of the Onion Plant: VI. A SENSITIVE MORPHOLOGICAL TEST FOR BULBING AND ITS USE IN DETECTING BULB DEVELOPMENT IN STERILE CULTURE

Bulb development in the onion plant, which occurs in responseto the stimulus of long days, has two main morphological features:swelling of the base of the pseudostem and the formation ofscales from leaf initials produced at the stem apex. The swellingmay be expressed as the ‘bulbing ratio’, i.e. theratio of the maximum diameter at the base to the minimum atthe neck, but this is a somewhat insensitive measure wider adverselight conditions and no ratio can be specified at which bulbingis first apparent. In the absence of bulbing, leaf initialshave at all stages of development a blade longer than the leafsheath, giving a ‘leaf ratio’ of blade/sheath atall times greater than unity. In scale formation, when bulbdevelopment begins, the sheath of a small initial starts togrow sooner than in foliage leaf formation and blade growthis suppressed; the leaf ratio therefore falls rapidly belowunity and this is diagnostic of bulbing. Characteristic graphsof leaf ratio plotted against leaf number from the apex demonstratethe stage of bulbing reached and a fall in leaf ratio from nearthe apex in such a graph can indicate the beginning of bulbingbefore any actual scales (with ratios less than unity) havebeen formed. In normally grown plants, differentiation intofoliage leaves or scales appears to take place when the initialsreach about 1 mm in length. When the incidence of bulbing within a treatment is very variable,difficulties arise in averaging the leaf ratio curves for thedifferent plants. Mean number of scales per plant, or mean minimumleaf ratio, may then be used as measures of bulbing. Heavy shading in winter was found to delay but not prevent thebulbing of those plants which survived. ‘Buds’, consisting of a portion of stem with theapex surrounded by leaf initials, but no scales, were dissectedout from onion sets and cultured aseptically in tubes. Limitedgrowth was obtained and rooting occurred in some cases. In thepresence of sucrose, swelling of leaf sheaths and reductionof the leaf ratios took place; these responses increased withsucrose concentration. Glucose similarly stimulated bulbingbut high osmotic pressure caused by mannitol or mineral nutrientshad no effect. Rooting appeared to reduce the degree of bulbing,possibly by competing for carbohydrate supply. The bulb developmentin these sterile cultures was most unusual in occurring in shortphotoperiods and the possibility of a residue of ‘bulbinghormone’ being stored in sets is discussed.     

Preparation of (+)-Trans-Isoalliin and Its Isomers by Chemical Synthesis and RP-HPLC Resolution

Naturally occurring (+)-trans-isoalliin, (R_CR_S)-(+)-trans-S-1-propenyl-L-cysteine sulfoxide, is a major cysteine sulfoxide in onion. The importance of producing it synthetically to support further research is very well recognized. The (+)-trans-isoalliin is prepared by chemical synthesis and reversed-phase (RP)-HPLC. First, S-2-propenyl-L-cysteine (deoxyalliin) is formed from L-cysteine and allyl bromide, which is then isomerized to S-1-propenyl-L-cysteine (deoxyisoalliin) by a base-catalyzed reaction. A mixture of cis and trans forms of deoxyisoalliin is formed and separated by RP-HPLC. Oxidation of the trans form of deoxyisoalliin by H₂O₂ produces a mixture of (−)- and (+)-trans-isoalliin. Finally, RP-HPLC is used successfully in separating (−)- and (+)-trans-isoalliin, and hence, (+)-trans-isoalliin is synthesized for the first time in this study. In addition, the (±) diastereomers of cis-isoalliin are also separated and purified by RP-HPLC.     

Bulb Development in Onion (Allium cepa L.) III. Effects of the Size of Adjacent Plants, Shading by Neutral and Leaf Filters, Irrigation and Nitrogen Regime and the Relationship between the Red: Far-red Spectral Ratio in the Canopy and Leaf Area Index

Field experiments were done to investigate why onion crops witha high leaf area index (LAI) initiate bulb scales and maturesooner than those with a low LAI. When small plants were growneither mixed with large plants or in pure stands, those in themixed stands initiated bulb scales earlier than those in purestands. The timing of bulb development therefore depended onthe size of neighbouring plants and the LAI of the stand andwas not simply determined by the size or age of individual plants.Shading plots with neutral filters which caused no spectralchanges slightly accelerated bulb development compared to unshadedplots but shading by a canopy of climbing pea and bean leaveshad a larger effect. Lack of irrigation advanced bulb maturitybut in the highly irrigated treatments plots of high plant densitystill initiated bulb scales and matured before those of lowdensity. Quantitative relationships between the change in R : FR andeither LAI or total radiation interception were determined foronion leaf canopies. It is suggested that the decrease in thered to far-red spectral ratio (R : FR) as LAI increases maybe the cause of the earlier bulb scale initiation and maturitythat occurs as LAI increases. Onion, Allium cepa (L.), spacing, competition, leaf area index, red: far red, irrigation, nitrogen, shading, bulbing     

Sulfur and nitrogen fertility affects flavour of field-grown onions

Although glasshouse studies have conclusively demonstrated that S nutrition can affect onion (Allium cepaL.) pungency this has been rarely observed in field-based studies due to difficulties in controlling S nutrition and lack of efficient methods for measurement of flavour bioactives. We have developed a rapid automated method for determination of onion lachrymatory factor ((Z, E)-thiopropanal-S-oxide; LF) based on juice extraction into dichloromethane and gas chromatography (GC) analysis with flame photometric detection. We evaluated this in a field trial of a mild (cv. ‘Encore’) and a pungent (cv. ‘Kojak’) onion cultivar grown on a low S soil with and without S addition, under high or low N treatments. No treatments significantly affected bulb fresh weight but S fertilisation significantly increased bulb total S, sulfate, pungency, LF and flavour precursor levels in both varieties. Analysis of bulb flavour precursors by HPLC confirmed that juice LF levels paralleled levels of the flavour precursor S-1-propenyl cysteine sulfoxide. The pungent cultivar also exhibited significant N main effects on bulb LF, total S and sulfate. We also assayed the key S-assimilatory enzyme, APS reductase (APR) in leaves before and during bulbing. Specific activities in the range of 1 to 11 nmol mg⁻¹·min⁻¹ were observed in youngest leaves, but only the milder cultivar exhibited significant stage × N × S effects. These findings suggest that sulfur metabolism of mild and pungent onions respond differently to N fertility, and that GC of LF is practical for field-based studies of onion flavour.     

Identification and characterization of novel senescence-associated genes from barley (Hordeum vulgare) primary leaves

Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence-specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up-regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence-associated expression was confirmed by Northern analyses or quantitative RealTime-PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence-induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1-like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF-like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1-GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed.     

HETEROPHYLLIC DEVELOPMENT IN MUEHLENBECKIA (POLYGONACEAE)

Flowering shoots of Muehlenbeckia platyclados Meisn. bear only reduced scale leaves which resemble the membranous sheath portion (ochrea) of leaves of other members of the Polygonaceae. Shoots propagated from cuttings bear enlarged foliage leaves with distinct lamina, petiole, and ochrea zones. The developmental basis for this heterophylly is explored in order to determine whether scale leaves resemble foliage leaves in their pattern of ontogeny or are developmentally unique. SEM and histological analyses have shown that scale leaves and foliage leaves are distinctive from inception. The scale leaf arises as a collarlike growth and extends over the shoot apex as a hooded sheath without evidence of blade initiation. By contrast, the first stage of foliage-leaf ontogeny is the differentiation of the distal lamina from the future leaf base. As the foliage-leaf ochrea encircles the stem axis, the lamina grows erect and projects from the abaxial surface of the sheath. Lamina reduction coupled with ochrea elaboration in intermediate leaf types indicate a homology between the entire scale leaf and foliage-leaf ochrea. Despite this homology, the production of the bladeless scale leaf does not involve a mere suppression of the foliage-leaf lamina. Erect growth of the saccate ochrea of the foliage leaf contrasts with the hooded expansion of the scale. Early histological differences, including contrasting rates of cell differentiation, also distinguish the two organs. This disparity in modes of growth and differentiation from inception results from separate, predetermined courses of ontogeny. Unlike other plants studied, leaf size and degree of leaf elaboration decrease with shoot meristem enlargement in Muehlenbeckia. Leaf packing does increase with shoot development and may contribute to variations in leaf morphology. It is concluded that the peculiarities of the heterophyllic leaf sequence in Muehlenbeckia are a property of the shoot system as a whole.     

Acclimation of photosynthesis to elevated CO2 in onion (Allium cepa) grown at a range of temperatures

Onion (Allium cepa) was grown in the field within temperature gradient tunnels (providing about ‐2.5°C to +2.5°C from outside temperatures) maintained at either 374 or 532 μmol mol^?1 CO₂. Plant leaf area was determined non‐destructively at 7 day intervals until the time of bulbing in 12 combinations of temperature and CO₂ concentration. Gas exchange was measured in each plot at the time of bulbing, and the carbohydrate content of the leaf (source) and bulb (sink) was determined. Maximum rate of leaf area expansion increased with mean temperature. Leaf area duration and maximum rate of leaf area expansion were not significantly affected by CO₂. The light‐saturated rates of leaf photosynthesis (A_sat) were greater in plants grown at normal than at elevated CO₂ concentrations at the same measurement CO₂ concentration. Acclimation of photosynthesis decreased with an increase in growth temperature, and with an increase in leaf nitrogen content at elevated CO₂. The ratio of intercellular to atmospheric CO₂ (C₁/C₃ ratio) was 7.4% less for plants grown at elevated compared with normal CO₂. A_sat in plants grown at elevated CO₂ was less than in plants grown at normal CO₂ when compared at the same C₁. Hence, acclimation of photosynthesis was due both to stomatal acclimation and to limitations to biochemical CO₂ fixation. Carbohydrate content of the onion bulbs was greater at elevated than at normal CO₂. In contrast, carbohydrate content was less at elevated compared with normal CO₂ in the leaf sections in which CO₂ exchange was measured at the same developmental stage. Therefore, acclimation of photosynthesis in fully expanded onion leaves was detected despite the absence of localised carbohydrate accumulation in these field‐grown crops.     

Pectic Enzyme Production by Fusarium oxysporum f. sp, ccpac: Induction by Cell Walls from Different Parts of Onion Bulbs at Different Growth Stages

Fusarium oxysporum f. sp. cepae produced significantly different amounts of pectic enzymes when grown on cell walls from morphologically different parts of onion bulbs. Cell walls from stem plate tissue of both tolerant and susceptible onion genotypes allowed a rapid and high production of both exo-polygalacturonase and endo-pectin-frans-eliminase. Bulb scale cell walls from susceptible genotypes induced synthesis of these enzymes at much lower rates and levels, whereas bulb scale cell walls from tolerant genotypes gave poor induction of pectic enzyme synthesis. Leaf sheath cell walls from both susceptible and tolerant genotypes were poor inducers of enzyme synthesis. Enzyme induction by cell walls from leaf sheaths and bulb scales of tolerant genotypes increased dramatically during ageing. Differences in pectic enzyme accumulation on cell walls were not related to fungal growth. These patterns of enzyme induction could help to explain susceptibility or tolerance of bulb scale and leaf sheath tissue of the different genotypes.     

Genotypic variation in sulphur assimilation and metabolism of onion (Allium cepa L.). II: Characterisation of ATP sulphurylase activity

To investigate the regulation of sulphur (S)-assimilation in onion further at the biochemical level, the pungent cultivar W202A and the milder cultivar Texas Grano 438 PVP (TG) have been grown in S-sufficient (S⁺; 4 meq S⁻¹) or S-deficient (S⁻; 0.1 meq S⁻¹) growth conditions, and tissues excised at the seedling stage (pre-bulbing; ca. 10-weeks-old) and at the mature stage (bulbing; ca. 16-weeks-old). S-supply negatively influenced adenosine-5′-phosphosulphate (APS) reductase (APR) enzyme activity in both cultivars at bulbing only, and a higher abundance of APR was observed in both cultivars at bulbing in response to low S-supply. In contrast, S-supply significantly influenced ATP sulphurylase (ATPS) activity in leaf tissues of W202A only, and only at bulbing, while an increase in abundance in response to high S-supply was observed for both cultivars at bulbing. To investigate the regulation of the ATPS enzyme activity and accumulation further, activity was shown to decrease significantly in roots at bulbing in the S-deficient treatment in both cultivars, a difference that was only supported by western analyses in W202A. Phylogenetic analysis revealed that AcATPS1 groups in a broad monocot clade with the closest sequences identified in Sorghum bicolour, Zea mays and Oryza sativa, but with some support for a divergence of AcATPS1. Detection of ATPS in leaf extracts after two dimensional gel electrophoresis (2-DE) revealed that the protein may undergo post-translational modification with a differential pattern of ATPS accumulation detected in both cultivars over the developmental progression from the seedling to the bulbing stage. Treatment of leaf extracts of W202A to dephosphorylate proteins resulted in the loss of immuno-recognised ATPS spots after 2-DE separation, although enzyme activity was not influenced. These results are discussed in terms of the tiers of control that operate at the biochemical level in the reductive S-assimilation pathway in a S-accumulating species particularly during the high-S-demanding bulbing stage.     

The Development of Chloroplast Ultrastructure and Hill Reaction Activity During Leaf Ontogeny in Different Maize (Zea Mays L.) Genotypes

Changes in Hill reaction activity (HRA) and ultrastructure of mesophyll cell (MC) chloroplasts were studied during the ontogeny of third leaf of maize plants using polarographic oxygen evolution measurement, transmission electron microscopy, and stereology. The chloroplast ultrastructure was compared in young (actively growing), mature, and senescing leaves of two different inbreds and their reciprocal F1 hybrids. Statistically significant differences in both HRA and MC chloroplast ultrastructure were observed between different stages of leaf ontogeny. Growth of plastoglobuli was the most striking characteristic of chloroplast maturation and senescence. The chloroplasts in mature and senescing leaves had a more developed system of thylakoids compared to the young leaves. Higher HRA was usually connected with higher thylakoid volume density of MC chloroplasts.     

Bulb Development in Onion (Allium cepa L.) I. Effects of Plant Density and Sowing Date in Field Conditions

Early and later maturing cultivars of both spring- and autumn-sownonions were sown on two dates at 25, 100 and400 plants m^–2.High levels of nutrients and irrigation were applied. Both high plant density and early sowing advanced the date atwhich bulb scales, rather than leaf blades, were initiated atthe shoot apex and so advanced the date of maturity by up to46 d. The later maturing spring-sown cultivar showed greaterresponses to density and sowing date than the earlier maturingspring-sown cultivar which in turn was more responsive thanthe autumn-sown cultivars. For each cultivar, maturity date increased linearly with decreasesin the percentage radiation intercepted by the leaf canopy. Onion, Allium cepa L., bulbing, competition, spacing     

The Development of Chloroplast Ultrastructure and Hill Reaction Activity During Leaf Ontogeny in Different Maize (Zea Mays L.) Genotypes

Changes in Hill reaction activity (HRA) and ultrastructure of mesophyll cell (MC) chloroplasts were studied during the ontogeny of third leaf of maize plants using polarographic oxygen evolution measurement, transmission electron microscopy, and stereology. The chloroplast ultrastructure was compared in young (actively growing), mature, and senescing leaves of two different inbreds and their reciprocal F1 hybrids. Statistically significant differences in both HRA and MC chloroplast ultrastructure were observed between different stages of leaf ontogeny. Growth of plastoglobuli was the most striking characteristic of chloroplast maturation and senescence. The chloroplasts in mature and senescing leaves had a more developed system of thylakoids compared to the young leaves. Higher HRA was usually connected with higher thylakoid volume density of MC chloroplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro bulb development in shallot (Allium cepa L. Aggregatum Group): effects of anti-gibberellins,sucrose and light

Bulbing was studied in shallot plants cultured in vitro. Bulbing occurred under a 16 h photoperiod with fluorescent + incandescent light and 30-50 g 1(-1) sucrose in the culture medium. Exogenous gibberellin (10 microM GA3) inhibited leaf and root growth and bulbing. When added to the medium at a concentration of 10 microM, three inhibitors of gibberellin biosynthesis (ancymidol, flurprimidol and paclobutrazol) promoted bulb formation and the percentage of bulbing. When ancymidol was used in combination with GA3, it did not reverse the effect of GA3 applied alone. Under treatments with 30-70 g l(-1) sucrose, bulbing ratios greater than those found in control plants were achieved by addition of ancymidol, and bulb fresh weight was increased in the same way. Ancymidol caused a 66% decrease in sucrose content in leaf bases but greatly increased the glucose, fructose and fructan contents. The increase in fructan content by ancymidol could result from the three-fold rise in total [14C]sucrose uptake per plant from the culture medium associated with a marked increase in leaf base labelling at the expense of root labelling. The possible role of ancymidol is discussed and evidence supports a major regulatory role for gibberellins in bulbing.