Gibberellins in Leaves of Alstroemeria hybrida: Identification and Quantification in Relation to Leaf Age

In alstroemeria (Alstroemeria hybrida), leaf senescence is retarded effectively by the application of gibberellins (GAs). To study the role of endogenous GAs in leaf senescence, the GA content was analyzed by combined gas chromatography and mass spectrometry. Five 13-hydroxy GAs (GA₁₉, GA₂₀, GA₁, GA₈, and GA₂₉) and three non-13-hydroxy GAs (GA₉ and GA₄) were identified in leaf extracts by comparing Kováts retention indices (KRIs) and full scan mass sprectra with those of reference GAs. In addition, GA₁₅, GA₄₄, GA₂₄, and GA₃₄ were tentatively identified by comparing selected ion monitoring results and KRIs with those of reference GAs. A number of GAs were detected in conjugated form as well. Concentrations of GAs in alstroemeria changed with the development of leaves. The proportion of biologically active GA₁ and GA₄ decreased with progressive senescence and the fraction of conjugated GAs increased. Received May 26, 1997; accepted August 12, 1997     

Gibberellin retards chlorophyll degradation during senescence of Paris polyphylla

Chlorophyll (Chl) degradation was found to be related to the endogenous gibberellin (GA) content in shoots during senescence in the perennial plant Paris polyphylla var. yunnanensis (Franch.). Treatment with gibberellic acid (GA₃) significantly increased the content of endogenous GAs (GA₄ + GA₇), retarded the senescence of shoots, and the degradation of proteins and Chl. Chlorophyllase, Mg-dechelation and peroxidase activities increased more in control plants than in those treated with GA₃. GA₃ treatment also protected lipoxygenase activity, which decreased significantly in control plants.     

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.     

NADP-Dependent Phenylacetaldehyde Dehydrogenase for Degradation of Phenylethylamine in Arthrobacter globiformis

The role of endogenous gibberellin (GA) in the flowering of the short-day plant, Pharbitis nil, was investigated by using uniconazole, which is a specific inhibitor of GA biosynthesis. Both the endogenous GA level and flowering response decreased with increasing concentration of uniconazole applied via the roots. The strongest inhibition of flowering was observed when uniconazole was applied one day before a 15-h dark treatment. The inhibition by uniconazole was overcome by an application of GAs to the plumules, the order of effectiveness of the endogenous GAs in P. nil being GA₁ ≧GA₂₀>GA₁₉≧GA₄₄>GA₅₃»GA_H. This is the first report of the correlation between the endogenous GA level and flowering response in P. nil. It was found that endogenous GAs were required for the flowering of P. nil during or just after the dark period.     

Changes in endogenous growth regulators in nasturtium leaves during senescence

Summary By use of lettuce-hypocotyl and wheat-coleoptile bioassay, the presence of both gibberellin (GA)-like and abscisic-acid(ABA)-like components in acidic ethyl-acetate extracts of fully expanded nasturtium (Tropaeolum majus) leaves has been shown. During senescence of detached leaves there was a progressive decline in GA-like components and an increase in ABA-like components. Pretreatment of detached leaves with GA₃ or kinetin prevented changes in the levels of endogenous growth regulators and delayed senescence. The observations provide experimental verification for the concept that senescence is associated with changes in endogenous growth regulators.     

Endogenous gibberellin and abscisic Acid content as related to senescence of detached lettuce leaves

Levels of gibberillins (GAs) and of abscisic acid (ABA) in attached leaves of romaine lettuce (Lactuca sativa L.) declined as the leaf became older. The time course of changes in hormone levels, determined in detached lettuce leaves kept in darkness, revealed that a sharp decline in GAs accompanied by a moderate rise in ABA occurred before the onset of chlorophyll degradation. As senescence advanced, no GAs could be detected and a considerable rise of ABA was observed. A similar sequence of hormonal modifications, but more pronounced, was observed in the course of accelerated senescence induced by either Ethephon or water stress. When kinetin or GA₃ was applied to detached leaves, the loss of chlorophyll and the rise in ABA were reduced. Bound GAs were detected in senescent leaves. They were not found in the kinetin-treated leaves, which contained a relatively high level of free GAs. The results suggest that senescence in detached romaine lettuce leaves is connected with a depletion of free GAs and cytokinins, which is thereafter followed by a great surge in ABA.     

Gibberellin-induced delay of leaf senescence of Alstroemeria cut flowering stems is not caused by an increase in the endogenous cytokinin content

The effects of various chemically pure gibberellins and cytokinins on leaf yellowing of Alstroemeria were described. The loss of chlorophyll was measured both in leaves of cut flowering stems and in a model system consisting of detached leaf tips. It was demonstrated that plant growth substances affected chlorophyll loss in both systems to the same extent. Leaf senescence was delayed by various gibberellins and cytokinins. The results demonstrated that some of the gibberellins (GA₄ and GA₇) are far more effective in delaying chlorophyll loss than GA₃, which is commonly used as a postharvest treatment for Alstroemeria cut flowering stems. Immunoassays were used to demonstrate that the effect of gibberellins on leaf yellowing does not involve an increase in the endogenous cytokinin concentrations in the leaves as an intermediate step.Abbreviations GA gibberellin A - HPLC high performance liquid chromatography - GA_3Mc GA₃-methyl ester - ZR zeatin riboside - IPAR isopentenyl adenine riboside.     

Gibberellins do not act against abscisic acid in the regulation of bulb dormancy of Allium wakegi Araki

Abscisic acid (ABA) is involved in bulb dormancy of Alliumwakegi Araki. We examined the antagonistic role of gibberellins(GAs)against ABA in the regulation of this dormancy. The concentrations of ABA andGAs in the basal leaf sheaths or bulbs of A. wakegi cv.Kiharawase were investigated during growth in the field and postharveststorage.The concentration of ABA in the basal leaf sheaths began to increase about onemonth before they began to swell, reached a maximum shortly after bulbharvesting, and decreased during postharvest storage. The plants showed bulbdormancy accompanied with the change in ABA concentration. GA1,GA3, GA4, GA12, GA15, GA19, and GA20 were identified in the basal leaf sheaths of A. wakegi from Kovats retention indices (KRI) andfull-scan mass spectra by gas chromatography - mass spectrometry (GC-MS)analysis. The concentrations of all classes of GAs in the basal leaf sheathsestimated by the dwarf rice micro-drop assay increased transitorily shortlybefore they began to swell, and decreased rapidly during bulb development. Bulbdormancy had already been induced when the concentration of the GAs becamemaximum. All the GAs in the bulbs remained at a low level during postharveststorage, when bulbs were gradually released from dormancy. The concentrationsof GA1+3, GA4, GA15, and GA20 inthe bulbs increased after sprouting of the bulbs planted in moist vermiculite.Hence, the state of bulb dormancy is considered to be independent of the GAconcentrations of in the basal leaf sheaths or bulbs of A.wakegi.     

A Monoclonal Antibody Recognizing Nonderivative 13-Hydroxy Gibberellins and Their Glucosides

The production and characterization of a monoclonal antibody (MAb AB10) against GA3-glucoside as well as GA3 is described. MAb AB10 was derived from an immunogen in which human serum albumin (HSA) was linked to GA3 at carbon-3. This antibody showed high affinity for GA3-glucoside as well as for 13-hydroxy gibberellins (GA1, GA3, GAs, etc). The affinity of MAb AB10 for 13-hydroxy GAs was significantly reduced by methylation of the 7-oic acid but not by glycosylation of 3-hydroxyl group. Based on this antibody, both of competitive enzyme-linked immunosorbent assays (ELISAs) for GA3-glucoside and for GA3 were developed. These two ELISAs displayed linear detection ranges from 0.2 pmol to 20 pmol. Using these assays, the fluctuation of GA3-1ike and GA3-glucoside-like sub-stances in the leaves of Rurnex japonicus was investigated. The results indicated that the glycosylation of free GAs was connected with leaf senescence and that the function of 6-ben- zyl amino purine in retarding the leaf senescence was probably related to delaying the process of glycosylation of free GAs.     

Gibberellin Metabolism and Transport During Germination and Young Seedling Growth of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The role of gibberellins (GAs) during germination and early seedling growth is examined by following the metabolism and transport of radiolabeled GAs in cotyledon, shoot, and root tissues of pea (Pisum sativum L.) using an aseptic culture system. Mature pea seeds have significant endogenous GA₂₀ levels that fall during germination and early seedling growth, a period when the seedling develops the capacity to transport GA₂₀ from the cotyledon to the shoot and root of the seedling. Even though cotyledons at 0–2 days after imbibition have appreciable amounts of GA₂₀, the cotyledons retain the ability to metabolize labeled GA₁₉ to GA₂₀ and express significant levels of PsGA20ox2 message (which encodes a GA biosynthesis enzyme, GA 20-oxidase). The large pool of cotyledonary GA₂₀ likely provides substrate for GA₁ synthesis in the cotyledons during germination, as well as for shoots and roots during early seedling growth. The shoots and roots express GA metabolism genes (PsGA3ox genes which encode GA 3-oxidases for synthesis of bioactive GA₁, and PsGA2ox genes which encode GA 2-oxidases for deactivation of GAs to GA₂₉ and GA₈), and they develop the capacity to metabolize GAs as necessary for seedling establishment. Auxins also show an interesting pattern during early seedling growth, with higher levels of 4-chloro-indole-3-acetic acid (4-Cl-IAA) in mature seeds and higher levels of indole-3-acetic acid (IAA) in young root and shoot tissues. This suggests a changing role for auxins during early seedling development.     

Distribution of endogenous gibberellins in vegetative and reproductive organs of Brassica

Recognizing the physiological diversity of different plant organs, studies were conducted to investigate the distribution of endogenous gibberellins (GAs) in Brassica (canola or oilseed rape). GA₁ and its biosynthetic precursors, GA₂₀ and GA₁₉, were extracted, chromatographically purified, and quantified by gas-chromatography-selected ion monitoring (GC-SIM), using [²H₂]GAs as internal standards. In young (vegetative) B. napus cv. Westar plants, GA concentrations were lowest in the roots, increased acropetally along the shoot axis, and were highest in the shoot tips. GA concentrations were high but variable in leaves. GA₁ concentrations also increased acropetally along the plant axis in reproductive plants. During early silique filling, GA₁ concentrations were highest in siliques and progressively lower in flowers, inflorescence stalks (peduncles plus pedicels), stem, leaves, and roots. Concentrations of GA₁₉ and GA₂₀ showed similar patterns of distribution except in leaves, in which concentrations were higher, but variable. Immature siliques were qualitatively rich in endogenous GAs and GA₁, GA₃, GA₄, GA₈, GA₉, GA₁₇, GA₁₉, GA₂₀, GA₂₄, GA₂₉, GA₃₄, GA₅₁, and GA₅₃ were identified by GC-SIM. In whole siliques, GA₁₉, GA₂₀, GA₁, and GA₈ concentrations declined during maturation due to declining levels in the maturing seeds; their concentrations in the silique coats remained relatively constant and low. These studies demonstrate that GAs are differentially distributed in _Brassica with a general pattern of acropetally increasing concentration in shoots and high concentration in actively growing and developing organs.     

识别未衍生化的13—羟化GAs及其葡萄糖苷的单克隆抗体

抗ＧＡ３ 及其葡萄糖苷的ＭＡＢ１０单克隆抗体源于以ＧＡ３ 中的３ 位羟基（３－ＯＨ）为偶联位点,人血清白蛋白（ＨＳＡ）为载体合成的ＧＡ３－３－ＨＳＡ 免疫原． 该抗体对１３－羟化ＧＡｓ（１３－ＯＨＧＡｓ、ＧＡ１、ＧＡ３、ＧＡ５ 等）和ＧＡ３ 葡萄糖苷具有高亲和力． ７ 位羧基的甲酯化可显著降低ＭＡＢ１０ 对１３－ＯＨ ＧＡｓ的亲和力,而３－ＯＨ 的糖苷化却未降低其亲和力． 用该抗体建立的两种分别用于ＧＡ３ 及其葡萄糖苷测定的酶联免疫吸附法（ＥＬＩＳＡ）,其检测线性范围均为０．２～２０ ｐｍ ｏｌ． 借助这两种ＥＬＩＳＡｓ,研究了羊蹄（Ｒｕｍ ｅｘ ｊａｐｏｎｉｃｕｓ）叶片中ＧＡ３ 及其类似ＧＡｓ和葡萄糖苷的动态变化．结果表明,叶片衰老与游离态ＧＡｓ的糖苷化有关;而６－ＢＡ 延缓衰老则可能与其减缓ＧＡｓ的糖苷化有关     

Elevated CO2 and leaf shape: Are dandelions getting toothier?

Heteroblastic leaf development in Taraxacum officinale is compared between plants grown under ambient (350 ppm) vs. elevated (700 ppm) CO₂ levels. Leaves of elevated CO₂ plants exhibited more deeply incised leaf margins and relatively more slender leaf laminae than leaves of ambient CO₂ plants. These differences were found to be significant in allometric analyses that controlled for differences in leaf size, as well as analyses that controlled for leaf developmental order. The effects of elevated CO₂ on leaf shape were most pronounced when plants were grown individually, but detectable differences were also found in plants grown at high density. Although less dramatic than in Taraxacum, significant effects of elevated CO₂ on leaf shape were also found in two other weedy rosette species, Plantago major and Rumex crispus. These observations support the long-standing hypothesis that leaf carbohydrate level plays an important role in regulating heteroblastic leaf development, though elevated C0₂ may also affect leaf development through direct hormonal interactions or increased leaf water potential. In Taraxacum, pronounced modifications of leaf shape were found at CO₂ levels predicted to occur within the next century.     

Effects of cytokinin and senescence-inducing factors on expression of P ARR5 -GUS gene construct during leaf senescence in transgenic Arabidopsis thaliana plants

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P _ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10⁻⁶ M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.     

Increased cytokinin levels in transgenic PSAG12–IPT tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis and N partitioning

We studied the impact of delayed leaf senescence on the functioning of plants growing under conditions of nitrogen remobilization. Interactions between cytokinin metabolism, Rubisco and protein levels, photosynthesis and plant nitrogen partitioning were studied in transgenic tobacco (Nicotiana tabacum L.) plants showing delayed leaf senescence through a novel type of enhanced cytokinin syn‐thesis, i.e. targeted to senescing leaves and negatively auto‐regulated (P_SAG12–IPT), thus preventing developmental abnormalities. Plants were grown with growth‐limiting nitrogen supply. Compared to the wild‐type, endogenous levels of free zeatin (Z)‐ and Z riboside (ZR)‐type cytokinins were increased up to 15‐fold (total ZR up to 100‐fold) in senescing leaves, and twofold in younger leaves of P_SAG12–IPT. In these plants, the senescence‐associated declines in N, protein and Rubisco levels and photosynthesis rates were delayed. Senescing leaves accumulated more (¹⁵N‐labelled) N than younger leaves, associated with reduced shoot N accumulation (–60%) and a partially inverted canopy N profile in P_SAG12–IPT plants. While root N accumulation was not affected, N translocation to non‐senescing leaves was progressively reduced. We discuss potential consequences of these modified sink–source relations, associated with delayed leaf senescence, for plant productivity and the efficiency of utilization of light and minerals.     

Cytokinin-Regulated Mesophyll Cell Division and Expansion during Development of Cucurbita pepo Leaves

The role of cytokinins in the development of mesophyll structure was studied in developing pumpkin Cucurbita pepo L. leaves. Leaves were treated with cytokinins at different stages of growth: when they reached 25 or 50% of their final size (S _max), immediately after leaf growth ceased, and during senescence. At the early stages of leaf development, treatment with exogenous benzyladenine accelerated division of mesophyll cells. At the later stages of development, BA treatment activated expansion of growing cells and those, which have just accomplished their growth. The exogenous cytokinin did not affect the senescent leaf cells. The content of endogenous cytokinins changed during mesophyll development. The juvenile leaves (25% of S _max) were characterized by low level of these phytohormones. In the expanding leaves (50% of S _max), the content of phytohormones increased and decreased when leaf growth ceased. In the senescent leaves, the cytokinin content decreased markedly. It was concluded that the response of mesophyll cells to cytokinin depended on the cell growth phase at the moment of hormone action. Furthermore, in the young leaves, lower cytokinin concentrations were required for division of mesophyll cells in vivo than for cell expansion at the final stage of leaf development.     

Assessing Gibberellins Oxidase Activity by Anion Exchange/Hydrophobic Polymer Monolithic Capillary Liquid Chromatography-Mass Spectrometry

Bioactive gibberellins (GAs) play a key regulatory role in plant growth and development. In the biosynthesis of GAs, GA3-oxidase catalyzes the final step to produce bioactive GAs. Thus, the evaluation of GA3-oxidase activity is critical for elucidating the regulation mechanism of plant growth controlled by GAs. However, assessing catalytic activity of endogenous GA3-oxidase remains challenging. In the current study, we developed a capillary liquid chromatography – mass spectrometry (cLC-MS) method for the sensitive assay of in-vitro recombinant or endogenous GA3-oxidase by analyzing the catalytic substrates and products of GA3-oxidase (GA₁, GA₄, GA₉, GA₂₀). An anion exchange/hydrophobic poly([2-(methacryloyloxy)ethyl]trimethylammonium-co-divinylbenzene-co-ethylene glycol dimethacrylate)(META-co-DVB-co-EDMA) monolithic column was successfully prepared for the separation of all target GAs. The limits of detection (LODs, Signal/Noise = 3) of GAs were in the range of 0.62–0.90 fmol. We determined the kinetic parameters (K _m) of recombinant GA3-oxidase in Escherichia coli (E. coli) cell lysates, which is consistent with previous reports. Furthermore, by using isotope labeled substrates, we successfully evaluated the activity of endogenous GA3-oxidase that converts GA₉ to GA₄ in four types of plant samples, which is, to the best of our knowledge, the first report for the quantification of the activity of endogenous GA3-oxidase in plant. Taken together, the method developed here provides a good solution for the evaluation of endogenous GA3-oxidase activity in plant, which may promote the in-depth study of the growth regulation mechanism governed by GAs in plant physiology.     

The leaf as the site of gibberellin action in flower formation in Bryophyllum daigremontianum

Summary The long-short-day plant Bryophyllum daigremontianum can be induced to flower by transfer from long to short days (LDSD), or by gibberellin (GA) application under SD. Application of GA to mature leaves of intact or partially defoliated plants induces flowering more effectively than when applications are made to the youngest leaf pair and the shoot tip.Mature leaves on de-budded plants in SD are induced to produce floral stimulus by GA application, as demonstrated by grafting LD receptor scions onto the debudded plants, or by grafting SD leaves treated with GA onto receptor stocks in LD. This shows that GA applied to Bryophyllum in SD exerts its flower-promoting effect in the leaves.The minimal number of SD necessary for flower formation in Bryophyllum is approximately 15, both in case of photoinduction by the shift LDSD, and after GA treatment in SD. It is concluded that the LD part of photinduction establishes a high level of endogenous GAs in the leaves which is a prerequisite for production of floral stimulus under subsequent SD.Work supported by the United States Atomic Energy Commission, Contract No. AT(11-1) 1338.