Juvenile-Specific Localization and Accumulation of a Rhamnosyltransferase and Its Bitter Flavonoid in Foliage,Flowers, and Young Citrus Fruits

1-2-Rhamnosyltransferase catalyzes the production of disaccharide-flavonoids that accumulate to 75% of dry weight. Vast energy is expended in a short time span to produce these flavonoids. The highest rhamnosyltransferase activities and immunodetected concentrations were observed in early development of Citrus grandis (pummelo), coinciding with up to 13% of fresh weight as naringin. The concentration of naringin in leaves, petals, receptacles, filaments, albedo, and flavedo drops drastically during development and correlates directly with a decrease in the activity and amounts of 1-2-rhamnosyltransferase. Anthers had minute rhamnosyltransferase activities and low concentrations of naringin. Conversely, high 1-2-rhamnosyltransferase activity and naringin concentrations appeared in both young and mature ovaries, as well as in young fruits. The total amounts of naringin in mature leaves decreased without detectable in vitro degradation of naringin in leaves. There was still a net accumulation of naringin in the albedo and flavedo of older fruit even though these tissues had only traces of 1-2-rhamnosyltransferase. Traces of enzyme synthesis in fruits, or import of the product from leaves, may explain the net accumulation of naringin in growing fruits. Unlike the late-expressed genes for glycosyltransferases in anthocyanin biosynthesis, the rhamnosyltransferases from Citrus are active only in juvenile stages of development.     

Bioproduction of neohesperidin and naringin in callus cultures of Citrus aurantium

The accumulation of both neohesperidin and naringin as major flavonoids in callus cultures of bitter orange (Citrus aurantium) was demonstrated using high performance liquid chromatography with a diode-array detector. The identity of both compounds was confirmed by their corresponding nuclear magnetic resonance spectra. The levels of neohesperidin are higher than those of naringin in callus culture, as they are in immature fruit, and high concentrations of both are found in young tissues such as immature fruits and the outer zone of calli.Abbreviations DMSO dimethylsulphoxide - DW dry weight - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - Rt retention time - V/UV visible/ultraviolet     

Anti-herbivore structures of Paulownia tomentosa: morphology, distribution, chemical constituents and changes during shoot and leaf development

Background and Aims: Recent studies have shown that small structures on plant surfacesserve ecological functions such as resistance against herbivores.The morphology, distribution, chemical composition and changesduring shoot and leaf development of such small structures wereexamined on Paulownia tomentosa. Methods: The morphology and distribution of the structures were studiedunder light microscopy, and their chemical composition was analysedusing thin-layer chromatography and high-performance liquidchromatography. To further investigate the function of thesestructures, several simple field experiments and observationswere also conducted. Key Results: Three types of small structures on P. tomentosa were investigated:bowl-shaped organs, glandular hairs and dendritic trichomes.The bowl-shaped organs were densely aggregated on the leavesnear flower buds and were determined to be extrafloral nectarines(EFNs) that secrete sugar and attract ants. Nectar productionof these organs was increased by artificial damage to the leaves,suggesting an anti-herbivore function through symbiosis withants. Glandular hairs were found on the surfaces of young and/orreproductive organs. Glandular hairs on leaves, stems and flowerssecreted mucilage containing glycerides and trapped small insects.Secretions from glandular hairs on flowers and immature fruitscontained flavonoids, which may provide protection against someherbivores. Yellow dendritic trichomes on the adaxial side ofleaves also contained flavonoids identical to those secretedby the glandular hairs on fruits and flowers. Three specialtypes of leaves, which differed from the standard leaves inshape, size and identity of small structures, developed nearyoung shoot tips or young flower buds. The density of smallstructures on these leaf types was higher than on standard leaves,suggesting that these leaf types may be specialized to protectyoung leaves or reproductive organs. Changes in the small structuresduring leaf development suggested that leaves of P. tomentosaare primarily protected by glandular hairs and dendritic trichomesat young stages and by the EFNs at mature stages. Conclusions: The results indicate that P. tomentosa protects young and/orreproductive organs from herbivores through the distributionand allocation of small structures, the nature of which dependson the developmental stage of leaves and shoots.     

Three new flavonoids,proanthocyanidin, and accompanying phenolic constituents from Feijoa sellowiana

Our investigation of phenolic constituents of fruits, flower buds, and leaves of Feijoa sellowiana led to the isolation of twenty-one phenolics including three new gossypetin glycosides 1–3, and also the purification of a proanthocyanidin fraction. A high-performance liquid chromatography method for simultaneous analysis of phenolic constituents was established and then used to investigate the phenolic profiles of the parts of the plant species, to show the presence of characteristic flavonoids and ellagic acid derivatives or ellagitannins in the extracts from fruits, flower buds, and leaves. The branch extract profile also suggested the presence of alkylated ellagic acids as characteristic constituents. Inhibitory effects of feijoa flavonoids on mushroom tyrosinase were seen, although in some cases this may have resulted from direct interaction with the enzyme. Cytotoxic effect of the proanthocyanidin fraction was also shown.     

High-performance liquid chromatography methods for the analysis of adrenergic amines and flavanones in Citrus aurantium L. var. amara

Reverse-phase HPLC coupled with photodiode array detection was used for the simultaneous separation and determination of naturally occurring adrenergic amines (octopamine, synephrine and tyramine) in fruits and dry extracts of Citrus aurantium L. var. amara and in herbal medicines derived therefrom. Synephrine was the main component in fruits (0.10-0.35%) and in dry extracts (3.00-3.08%) and was present in the range 0.25-0.99% in herbal medicines. Flavanones were analysed in the same samples using a reverse-phase HPLC technique which allowed the identification and quantification of neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, naringenin and hesperetin. C. aurantium fruits and derivatives contained mainly glycosylated flavanones: in particular, naringin and neohesperidin were found to be the major flavonoids and their concentrations ranged from 1.80 to 26.30 and from 3.90 to 14.71 mg/g, respectively. The levels of aglycones were very low in all samples tested.     

Invertase Activity, Soluble Carbohydrates and Inflorescence Development in the Tomato (Lycopersicon esculentum Mill.)

The concentration of reducing sugars in the developing firstinflorescence of the tomato (Lycopersicon esculentum Mill.)increased steadily between the macroscopic appearance of theflower buds and the initial stages of fruit expansion. Overthis period sucrose concentrations remained relatively constant.The rise in reducing sugar concentration was accompanied byan increase in the activity of an acid invertase. In individualflower buds invertase activity rose to a maximum shortly beforeanthesis and declined sharply as the anthers dehisced. Increased planting densities and removal of source leaves reducedthe rate of dry matter accumulation by the first inflorescenceand increased the incidence of flower bud abortion. These changeswere correlated with reductions in reducing sugar concentrations,in reducing sugar/sucrose ratios and in acid invertase levels.Removal of young leaves at the shoot apex significantly increasedthe relative growth rate of the inflorescence and led to a substantialincrease in its invertase content. These treatments had relativelylittle effect on sucrose concentration in the inflorescence. The data are consistent with the operation of an invertase-mediatedunloading mechanism for transported sucrose at sinks in theflower buds. It is suggested that the retarded development ofthe first inflorescence and the high incidence of flower budabortion observed under conditions of reduced photoassimilateavailability are causally related to the decline in invertaseproduction in the flower buds. Possible mechanisms for the regulationof invertase synthesis in the flowers are discussed. Lycopersicon esculentum Mill, tomato, inflorescence development, invertase, sink activity     

The role of endogenous growth regulators in the differentiation processes of walnut (Juglans regia L.)

The character of endogenous growth substances was investigated in developing buds, young fruits and mature walnut leaves. The relatively high content of auxins and gibberellin-like substances was found by means of bioassays in the youngest primordia of vegetative buds. The level of auxins drops in the further course of primordia transformation into the staminate catkins. The development of leaf-buds is characterized by the accumulation of inhibitory activity as revealed by theAvena bioassay, whereas the data obtained from the lettuce bioassay indicate a pronounced stimulation. The onset of terminal bud development is also accompanied by inhibitions and it is only with pistillate flower differentiation that the temporary rise in auxin level is observed. An inhibitory activity was found in these extracts using lettuce bioassay. There is a relatively high auxin level in young fruits, mature leaves and resting buds during the mid-summer period whereas the accumulation of clearcut inhibitions is signalled by the results of lettuce bioassay. The regulatory role of growth substances in differentiation may be better understood during the second year as many leaf-abnormities appear only with the outgrowing of the bud. Abnormal catkins differ in the number of florets and stamens and some even bear pistillate flowers. Fruit development is liable to deviations in the early stages of differentiation. Abnormal fruits enable us to elucidate many structural peculiarities.     

Flavonoids in Citrus and Related Genera

The occurence and distribution of flavonoid glycosides in young leaves and young and mature fruits of many citrus species and trifoliate orange were investigated. The occurence of neohesperidin in both young leaves and young fruits is fairly common to a number of species in subgenus Archicitrus. Ripe fruits of citrus could be classified into (a) the hesperidin group (b) the neohesperidin group (c) the naringin group and (d) the isonaringin group. A new flavanone glycoside, isonaringin, isolated from young fruits of Jagatarayu and Teng mikan is slightly bitter and has been determined by chemical and spectral evidences to have the structure of naringenin-7-rhamnoglucoside. Data showing the occurence of flavanone glycosides in some artificial citrus hybrids were also given.     

Floral determination in the terminal and axillary buds of Nicotiana tabacum L

The terminal and axillary buds of the day-neutral plant, Nicotiana tabacum cv. Wisconsin 38, become determined for floral development during the growth of the plant. This state of determination can be demonstrated with a simple experiment: buds determined for floral development produce the same number of nodes in situ and if rooted. After several months of growth and the production of many leaves, the terminal bud became determined for floral development within a period of about 2 days. After the terminal bud became florally determined, it produced four nodes and a terminal flower. The buds located in the axils of leaves borne just below the floral branches became florally determined 5 to 9 days after the terminal bud became florally determined. Since florally-determined axillary buds were not clonally derived from a florally-determined terminal meristem, axillary buds and the terminal bud acquired the state of floral determination independently. These data indicate that a pervasive signal induced a state of floral determination in competent terminal and axillary buds.     

Flavonoids in Citrus Hybrids

The occurrence and distribution of flavanone glycosides in the leaves and fruits of many kinds of artificial citrus hybrid plants were investigated by polyamide thin-layer chromatography. The citrus hybrids can be divided into two broad categories, a) those containing rutinosyl glycosides, b) those containing neohesperidosyl glycosides in accordance with the case of natural citrus species. The fiavonoid patterns of rutinosyl glycosides are classified into the following groups, a) hesperidin, b) narirutin, c) hesperidin and narirutin, d) didymin and narirutin, e) hesperidin, narirutin and eriocitrin and f) hesperidin and eriocitrin, while the pattern of neohesperidosyl glycosides fall into six groups, a) naringin, b) neohesperidin and naringin, c) neohesperidin, naringin and neoeriocitrin, d) neohesperidin and neoeriocitrin, e) naringin and neoeriocitrin, and f) poncirin, neohesperidin, naringin and neoeriocitrin. It is worthy of note that a hybrid (accession number 1088) between C. unshiu and C. hassaku contains only narirutin. Among the ninty-four hybrids examined, fifty-three varieties were obviously different from female parents in their flavonoid pattern and could be judged as true hybrids by fiavonoids but the others could not.

Additionally, a survey of fiavonoids in newly found natural pummelo- and Daidai hybrids were carried out in connection with their origin.     

Composition of the essential oils in various organs at different developmental stages of Ammi visnaga (L.) Lam. from Tunisia

The composition of the essential oils isolated by hydrodistillation from various organs at different development stages of Ammi visnaga (L.) Lam. growing in Tunisia was determined by GC/MS analysis. In particular, the oil profiles of the leaves, stems, flower buds, roots, umbels, and fruits have been examined during the whole life cycle. The oil from the flowering aerial parts was characterized by a high content of isoamyl 2-methylbutanoate. After flowering and during desiccation and fructification, the umbels and fruits expressed a high content of linalool. The oils, extracted from the roots collected in the vegetatif, buds floral, and floral stages, were rich in monoterpene aldehydes, oxygenated monoterpenes, and monoterpene hydrocarbons. The highest level of non-terpene hydrocarbons was found at the flower-bud stage, represented by 61.3% of nonane. Among the monoterpenes, sabinene (12.5%) and β-pinene (8.5%) were identified in the flower buds.     

Variations of Flavonoid Composition and Antioxidant Properties among Different Cultivars,Fruit Tissues and Developmental Stages of Citrus Fruits

A large number of biologically active compounds are present in ripe citrus fruits. However, few studies have been focused on the changes in flavonoids and the evolution of antioxidant activity during citrus fruit growth. In this study, fruits of five citrus cultivars cultivated in China were sampled at 60–210 days post‐anthesis (DPA) at intervals of 30 days. The amounts of main flavonoids in the peel and pulp were analyzed by HPLC and their activities were studied by DPPH, ABTS and FRAP. The results showed that the contents of hesperidin, diosmin, eriodictyol, rutin and nobiletin increased before 90 DPA and then decreased with the growth and development of fruits, but an opposite tendency was observed for naringin and narirutin. The antioxidant activities in citrus peel and pulp were found to be significantly correlated with some flavonoids. The results may be of guiding values in citrus production and utilization of citrus fruit by‐products.     

The role of free polyamines in the alternate-bearing of pistachio (<Emphasis Type="Italic">Pistacia vera</Emphasis> cv. Pontikis)

The present study was conducted in order to examine the physiological role of free polyamines in flower bud abscission. For this reason five 15-year old pistachio trees cv. "Pontikis" were selected and half of the main branches were manually defruited in early May. Polyamines were analyzed in three different organs (shoots, leaves and flower buds) from both fruiting and non-fruiting branches, during the period of kernel growing. Five samplings took place and the polyamines putrescine, spermidine and spermine were assayed. The flower bud abscission percentage was recorded every 5–10 days during kernel formation. Polyamine concentration declined during the period that coincides with that of kernel development, in both fruiting and non-fruiting branches, while significant bud abscission occurred from mid-July till late September in fruiting branches. Polyamine concentration in organs from fruiting branches was in most cases lower than that of non-fruiting ones. Most of the individual polyamines exhibited a high and significant negative correlation with bud abscission. By measuring the spermidine content of leaves and the spermine content of buds, it was possible to estimate the forthcoming bud abscission with significant accuracy (approximately 93%). On the other hand, the total polyamine content of the buds exhibited a significant strong negative relationship with bud abscission. Consequently, polyamines could have an important physiological function in the development of flower bud abscission of pistachio.     

Endogenous gibberellins and inhibitors in relation to flower induction and inflorescence development in the olive

Comparative analyses of reproductive and vegetative tissues of the olive (Olea europaea L. cv. Manzanillo) for endogenous hormones, particularly inhibitors and gibberellin like substances, were made to study the relation between such hormones and thermoinduction of flowering. Qualitative and quantitative changes in gibberellin-like subtance(s) were observed in lateral buds (potential flower buds) but not in leaves or terminal buds (potential vegetative buds) sampled from orchard trees at intervals during the winter and spring. At least two types of gibberellin-like substances were found in extracts of lateral buds; their levels increased progressively during the low temperature induction period, reaching a maximum shortly before floral initiation. Two types of inhibitors were extracted from buds and leaves. A nonacidic type did not change during the induction stage but decreased considerably during the initiation period. An acidic inhibitor, which was identified as an abscisic acid-like substance, was present at a relatively lower level in lateral (flower) buds than in terminal (vegetative) buds during the induction period.     

Naringin Levels in Citrus Tissues : II. Quantitative Distribution of Naringin in Citrus paradisi MacFad

The quantitative distribution of the flavanone-7-neohesperidoside, naringin, in seeds, seedlings, young plants, branches, flowers, and fruit of Citrus paradisi Macfad., cv `Duncan' was analyzed by radioimmunoassay. High levels of naringin were associated with very young tissue and lower levels were found in older tissues. Seed coats of ungerminated seeds and young shoots had high naringin concentrations whereas cotyledons and roots had very low concentrations. Light-grown seedlings contained nearly twice as much naringin as etiolated seedlings and, in young plants and branches, the naringin content was highest in developing leaves and stem tissue. In flowers, the ovary had the highest levels of naringin, accounting for nearly 11% of the fresh weight. There was a net increase in the total naringin content of fruits during growth. However, due to the large increase in fruit size, there was a concomitant decrease in the naringin concentration as the fruit matured.     

Cloning and characterisation of a pepper aquaporin,CaAQP, which reduces chilling stress in transgenic tobacco plants

Ubiquitous cell membrane proteins called aquaporins are members of major intrinsic proteins (MIPs), which control the specific transport of water molecules across cell membranes. A pepper aquaporin gene (CaAQP), which exhibits the structural features of tonoplast intrinsic proteins of the MIP subfamily, was isolated from the leaves of chilling-treated seedlings of pepper (Capsicum annuum L.) cv. P70. Assays indicated high levels of expression in young seeds, green fruits and flower buds and low levels of expression in the stems, leaves and roots of pepper. The expression patterns were strongly and rapidly induced by HgCl₂, low temperature, abscisic acid, fluridone and osmotic stresses. The responsiveness of pepper seedlings pretreated with abscisic acid at low temperatures demonstrated up-regulation of CaAQP by chilling, which is potentially involved in ABA signalling. Our results indicated that overexpression of CaAQP decreased chilling stress in transgenic plants, likely by increasing the stomatal aperture under stress, increasing the rate of membrane damage during the recovery stage, thereby affecting the intercellular CO₂ concentration with lower stomatal conductance and transpiration rates. VIGS of CaAQP in pepper plants caused significant growth retardation. These results suggested that CaAQP played a crucial role in the plant response to abiotic stresses.     

The biomass and nutritive potential of Vallisneria americana Michx in navigation pool 9 of the upper Mississippi river

Vallisneria americana Michx (wild celery) was studied to determine the biomass and nutritive potential of all morphological structures. A 2.6-ha stand of uniform V. americana was sampled during the summer and autumn of 1980, and the spring and summer of 1981 in the southern portion of Navigation Pool 9 of the Upper Mississippi River.The maximum production rate of 3.2 g m^?2 day^?1 was coincident with rapid rosette production and flowering, and occurred mid- to late-July 1980. The maximum biomass of 217.3 g dry wt. m^?2 was on 1 September 1980, when fruit development was also at a maximum. Leaves composed 60–70% of the summer biomass; winter buds constituted all of the winter biomass.Winter buds and fruits had the greatest nutritive potentials. Both organs contained relatively high dry matter concentrations and were low in ash (less than 10%) and fiber content. The potentially-digestible ash-free non-cell-wall fraction (NCF) was composed of an average of 75.7 and 82.2% of the dry weight of fruits and winter buds, respectively. In contrast, the nutritive potential of leaves, rootstocks, peduncles and stolons was reduced because of high moisture (less than 8% dry matter), ash and fiber concentrations. Staminate inflorescences and pistillate flowers were high in crude protein (averaged 21.8% and 16.1% of the dry-weight, respectively) and ash-free non-cell-wall fractions, but they accounted for only 2.7% of the plant biomass. The maximum calorific content of V. americana was approximately 3200 kJ m^?2 at peak biomass on 1 September 1980.     

Loss of flower bud vigour in the Mediterranean shrub,Cistus albidus L. at advanced developmental stages

To better understand aging in perennials, age‐related changes in the physiology of leaves and flower buds of the Mediterranean shrub, Cistus albidus L. were evaluated. Two groups of different ages (5 and 10 years old), both at advanced developmental stages but of similar size, were compared. Total plant biomass, biomass produced per apical meristem and levels of cytokinins, abscisic acid and jasmonic acid in leaves and flower buds, as well as flower production, were measured. No differences in plant size, vegetative growth rates and levels of phytohormones in leaves were observed between 5‐ and 10‐year‐old plants. However, they showed significant differences in flower bud development; the older plants having reduced vigour, with 29.6% of flowers reaching anthesis compared to 52.5% in the younger plants. Furthermore, endogenous concentrations of zeatin and abscisic acid in flower buds at stage I (start of flower organ formation) were 61% and 41%, respectively, smaller in 10‐ than in 5‐year‐old plants. At stage II (with all flower organs formed), zeatin and abscisic acid concentrations decreased by ca. 90% and 80%, respectively, but differences between age groups were still evident (60% and 29% for zeatin and abscisic acid, respectively). Jasmonic acid levels in flower buds decreased by 80% from stage I to II, but did not differ between age groups. Despite reductions in flower bud vigour, total number of flowers per individual was not significantly different between age groups, so that an age‐related loss in reproductive vigour at the organ level did not lead to a decrease in flower production at the whole plant level.