Study on the extraction, purification and quantification of jasmonic acid, abscisic acid and indole-3-acetic acid in plants

Introduction  – Jasmonic acid (JA), abscisic acid (ABA) and indole‐3‐acetic acid (IAA) are important plant hormones. Plant hormones are difficult to analyse because they occur in small concentrations and other substances in the plant interfere with their detection. Objective  – To develop a new, inexpensive procedure for the rapid extraction and purification of IAA, ABA and JA from various plant species. Methodology  – Samples were prepared by extraction of plant tissues with methanol and ethyl acetate. Then the extracts were further purified and enriched with C₁₈ cartridges. The final extracts were derivatised with diazomethane and then measured by GC‐MS. The results of the new methodology were compared with those of the Creelman and Mullet procedure. Results  – Sequential elution of the assimilates from the C₁₈ cartridges revealed that IAA and ABA eluted in 40% methanol, while JA subsequently eluted in 60% methanol. The new plant hormone extraction and purification procedure produced results that were comparable to those obtained with the Creelman and Mullet's procedure. This new procedure requires only 0.5 g leaf samples to quantify these compounds with high reliability and can simultaneously determine the concentrations of the three plant hormones. Conclusion  – A simple, inexpensive method was developed for determining endogenous IAA, ABA and JA concentrations in plant tissue. Copyright © 2008 John Wiley & Sons, Ltd.     

Estimation of endogenous contents of phytohormones during internode development in <Emphasis Type="Italic">Merremia emarginata</Emphasis>

During the entire period of internode growth of Merremia emarginata contents of gibberellic acid (GA₃), phenyl-acetic acid (PAA), indole-3-acetic acid (IAA, free and conjugated) and abscisic acid (ABA, free and conjugated) were estimated by ELISA using polyclonal antibodies raised against each hormones. At the time of internode elongation free auxin content was low and increased with the decrease in the rate of elongation. In contrast, conjugated IAA showed declining trend where free IAA content was remarkably high, suggesting thereby that conjugated IAA might have mobilized during the later phase of internode development. The endogenous GA₃ contents were high as compared to other hormones; however, no significant role of GA₃ was discernible in elongation growth. Conjugated ABA contents remained very low during the elongation growth and increased thereafter.     

扁桃生理落果期不同组织激素浓度的动态变化及其对落果的影响

为探明扁桃幼果生理脱落与GA3、IAA和ABA等3种激素的关系,以新疆‘纸皮’扁桃为试材,分析新梢、结果枝组、幼果和果柄(包括正常发育幼果和果柄、即将脱落幼果和果柄)中3种内源激素浓度的动态变化规律,并分别涂抹3种外源激素调查其对坐果率的影响。结果表明:(1)‘纸皮’扁桃幼果脱落期和新梢生长期重合,扁桃生理落果期不同组织中3种内源激素浓度变化趋势与新梢生长期和幼果脱落期的动态特征基本一致。(2)扁桃生理落果期间,正常的和即将脱落的幼果及其果柄中内源激素浓度呈规律性变化,即:GA3和IAA浓度表现为正常果和正常果果柄始终大于相应的落果和落果果柄,而内源ABA浓度表现则与之相反,同时对应外源涂抹试验也印证了幼果和幼果果柄中高GA3和IAA浓度、低ABA浓度有利于扁桃坐果。(3)新梢和幼果中对应内源激素之间的浓度平衡关系也是调控扁桃幼果生理脱落的重要因素,即:新梢与幼果的GA3比值和IAA比值增大、而ABA比值减少将会促进幼果脱落,反之则减缓幼果脱落。     

Plant growth hormone production from olive oil mill and alcohol factory wastewaters by white rot fungi

In this study, olive oil mill and alcohol factory wastewaters have been tested as growth media for the production of plant growth hormones. Funalia trogii ATCC 200800 and Trametes versicolor ATCC 200801 have been tested. Gibberellic acid (GA₃), abscisic acid (ABA), indole acetic acid (IAA), and cytokinin were determined in the culture media of these fungi. Both organisms produced enhanced levels of all three hormones in the presence of either of the wastewaters.     

Physiological characteristics and related gene expression of after‐ripening on seed dormancy release in rice

After‐ripening is a common method used for dormancy release in rice. In this study, the rice variety Jiucaiqing (Oryza sativa L. subsp. japonica) was used to determine dormancy release following different after‐ripening times (1, 2 and 3 months). Germination speed, germination percentage and seedling emergence increased with after‐ripening; more than 95% germination and 85% seedling emergence were observed following 1 month of after‐ripening within 10 days of imbibition, compared with <45% germination and 20% seedling emergence in freshly harvested seed. Hence, 3 months of after‐ripening could be considered a suitable treatment period for rice dormancy release. Dormancy release by after‐ripening is mainly correlated with a rapid decline in ABA content and increase in IAA content during imbibition. Subsequently, GA₁/ABA, GA₇/ABA, GA₁₂/ABA, GA₂₀/ABA and IAA/ABA ratios significantly increased, while GA₃/ABA, GA₄/ABA and GAs/IAA ratio significantly decreased in imbibed seeds following 3 months of after‐ripening, thereby altering α‐amylase activity during seed germination. Peak α‐amylase activity occurred at an earlier germination stage in after‐ripened seeds than in freshly harvested seeds. Expression of ABA, GA and IAA metabolism genes and dormancy‐related genes was regulated by after‐ripening time upon imbibition. Expression of OsCYP707A5, OsGA2ox1, OsGA2ox2, OsGA2ox3, OsILR1, OsGH3‐2, qLTG3‐1 and OsVP1 increased, while expression of Sdr4 decreased in imbibed seeds following 3 months of after‐ripening. Dormancy release through after‐ripening might be involved in weakening tissues covering the embryo via qLTG3‐1 and decreased ABA signalling and sensitivity via Sdr4 and OsVP1.     

Carbonic Anhydrase,Photosynthesis, and Seed Yield in Mustard Plants Treated with Phytohormones

The leaves of 30-d-old plants of Brassica juncea Czern & Coss cv. Varuna were sprayed with 10^–6 M aqueous solutions of indole-3-yl-acetic acid (IAA), gibberellic acid (GA₃), kinetin (KIN), and abscisic acid (ABA) or 10^–8 M of 28-homobrassinolide (HBR). All the phytohormones, except ABA, improved the vegetative growth and seed yield at harvest, compared with those sprayed with deionised water (control). HBR was most prominent in its effect, generating 32, 30, 36, 70, 25, and 29 % higher values for dry mass, chlorophyll content, carbonic anhydrase (E.C. 4.2.1.1) activity, and net photosynthetic rate in 60-d-old plants, pods per plant, and seed yield at harvest, over the control, respectively. The order of response to various hormones was HBR > GA₃ > IAA > KIN > control > ABA.     

不同成花量金花茶花果期果枝叶内源激素的变化

该研究采用ISSR分子标记,对黄枝油杉7个自然种群的遗传多样性进行了分析。结果表明：用12条ISSR引物对218个黄枝油杉个体进行扩增,共扩增出125个位点。在物种水平上,多态性位点百分数( PPL)为100.00％,Shannon信息多样性指数( I)为0.4177,Nei’ s基因多样性指数( H)为0.2666;在种群水平上,多态性位点百分数(PPL)在71.20％~92.00％之间,平均值为80.69％,Shannon信息多样性指数(I)在0.3273~0.3886之间,平均值为0.3548,Nei’ s基因多样性指数( H)在0.2139~0.2478之间,平均值为0.2291。这说明黄枝油杉在物种水平和种群水平上均显示出较高的遗传多样性。 Nei’ s遗传多样性分析( Gst＝0.1433)和AMOVA分析(Φst＝17.91％)表明,黄枝油杉的遗传变异主要存在于种群内,种群间的遗传分化程度较低,种群间保持一定的基因交流( Nm＝2.9890>1)。 Mantel分析显示,黄枝油杉种群间的遗传距离和地理距离之间不存在显著的相关关系( r＝0.4567, P＝0.0610>0.05)。     

Comparative Effects of lndole-3-acetic acid, Abscisic acid, Gibberellic acid and 6-Benzylaminopurine on the Dark- and Light-induced Pulvinar Movements in Cassia fasciculata Michx

Effects of plant hormones were examined on the dark- and light-inducedmovements of Cassia fasciculata. Indole-3-acetic acid (IAA),gibberellic acid (GA₃) and 6-benzylaminopurine (6-BAP) inhibitedthe scotonastic movement whereas abscisic acid (ABA) enhancedit. After brief treatments (5 to 30 min), the ABA effect wasinhibitory rather than promotional. Hormonal treatment in theacidic range gave the best physiological response for ABA, butthe greatest efficiency of IAA, GA₃ and 6-BAP was obtained withpH values close to neutrality. Three to 5 h were needed beforeexpression of the physiological effect triggered by GA₃ and6-BAP, while 5 min treatments were sufficient for IAA and ABA.Light-induced movements were largely enhanced by IAA and slightlyby GA₃ but inhibited by 6-BAP and ABA. The results are discussedin relation to the ionic changes in the pulvinar motor cells,regulating leaflet movements. Key words: Abscisic acid, auxins, cytokinins, gibberellic acid, pulvinar movements     

Induced parthenocarpy—a way of changing the levels of endogenous hormones in tomato fruits (Lycopersicon esculentum Mill.) 1. Extractable hormones

Ethylene, indol-3-acetic acid (IAA), gibberellin-like substances (GAs) and abscisic acid (ABA) were analysed in extracts from normal, seed-containing and parthenocarpic tomato fruits throughout fruit development. Parthenocarpic fruit growth was induced with an auxin (4-CPA), morphactin (CME) or gibberellic acid (GA₃) and compared with that of pollinated control fruits. Fruit growth was only affected by the treatment with GA₃, decreasing size and fresh weight by 60%. The peak sequence of hormones during fruit development was ethylene-GAs-IAA-ABA. Seeded fruits contained the highest levels of IAA and ABA but the lowest levels of GAs. Also, in seeded fruits, a high proportion of IAA and ABA was found in the seeds whereas this was not the case for GAs.Hormone levels of tomato fruits may be successfully, easily and reproducibly altered by inducing parthenocarpic fruit growth and thus eliminating development of seeds which are a major source of hormone synthesis. In spite of markedly changed hormone levels, there was no obvious relationship between fruit growth and extractable hormones per se. However, the results indicate that a high ratio of GAs: auxins is unfavourable for growth of tomato fruits.     

Induced parthenocarpy-a way of manipulating levels of endogenous hormones in tomato fruits (Lycopersicon esculentum Mill.) 2. Diffusible hormones

Indol-3-acetic acid (IAA), gibberellin-like substances (GAs), and abscisic acid (ABA) were measured throughout the first 35 days of fruit development in agar diffusates from seeded and parthenocarpic tomato fruits. Parthenocarpic fruit growth was induced with either an auxin (4-CPA), morphactin (CME) or gibberellic acid (GA₃). IAA and GAs were at their highest levels in diffusates during the early stages of fruit growth, whereas diffusible ABA increased later. Most IAA was found in diffusates from auxin-induced and seeded fruits, whereas GAs were at their lowest levels in seeded fruits. There were only minor differences in ABA concentrations regardlesss of the treatments.Levels of diffusible hormones of tomato fruits may be easily manipulated by inducing parthenocarpic fruit growth. In spite of no obvious relationship between fruit growth and hormone levels in this study, induced parthenocarpy is considered a useful tool to further elucidate the role of hormones in fruit development and sink-source interactions.     

The effect of plant growth regulator treatments on the levels of ethylene emanating from excised turgid and wilted wheat leaves

Abscisic acid (ABA) inhibits the production of ethylene induced by water stress in excised wheat leaves and counteracts the stimulatory effect of 6-benzyladenine (BA) on this process. The stimulatory effect of BA and the inhibitory effect of ABA were equally pronounced whether external or endogenous ethylene levels were determined. When leaves were sprayed or floated on solutions of BA, indole-3-acetic acid (IAA), gibberellic acid (GA₃), or ABA, the relative activities of these growth regulators on stress-induced ethylene at 10^-4 mol l^-1 were BA>IAA >GA₃>controls>ABA. In non-stressed leaves, however, where the levels of ethylene produced were 2–20 times smaller, the relative activities were IAA >BA>GA₃>controls>ABA. The effects of BA and ABA spray treatment on water stress induced ethylene were closely similar whether the solutions were applied 2 or 18 h prior to the initiation of water stress. The relationships between the levels of endogenous growth regulators in the plant and ethylene release induced by water stress are discussed.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - GA₃ gibberellic acid - ABA abscisic acid - GLC gas-liquid chromatography - _leaf leaf water potential     

Pollen tube growth is affected by exogenous hormones and correlated with hormone changes in styles in <Emphasis Type="Italic">Torenia fournieri</Emphasis> L.

In the present report, we described the effects of indole-3-acetic acid (IAA), zeatin (ZT), gibberellin (GA₃), and abscisic acid (ABA) on in vitro pollen germination and pollen tube growth in Torenia fournieri L. The results showed that IAA and GA₃ stimulated in vitro pollen tube growth, ABA inhibited pollen tube growth, and ZT had no significant effect on the process. The stimulating effect of exogenous IAA was particularly distinct, and led to synchronous growth of straighter and more slender pollen tubes compared with the controls. However, no significant changes were found in the germination of the treated pollen. The auxin efflux inhibitor, 10 μM 1-N-naphthylphthalamic acid (NPA), was also found to stimulate pollen tube growth. We measured the content of hormones (free IAA, ZT, GA₃, and ABA) in the stigmas and styles before and after pollination. The hormone contents of stigmas measured 0.5 h after pollination (0.5 HAP) showed that ABA content decreased, whereas the content of IAA, ZT, or GA₃ did not change significantly. The hormone level in pollinated styles (4 HAP) when pollen tubes had grown into the middle part of style was characterized by an increase in free IAA and GA₃ and a decrease in ABA, which was in agreement with the results that IAA and GA₃ promoted but ABA inhibited pollen tube growth in vitro. Furthermore, the change of IAA level in styles was most notable, which was accordant to the fact that auxin stimulated significantly pollen tube growth in vitro. Using immunoenzyme and immunogold labeling techniques and an anti-IAA monoclonal antibody, we confirmed that free IAA was present throughout style tissues, and distributed in the nucleus and cytoplasm of style cells. All these results suggested that hormones, especially IAA, play important roles in pollen tube growth of T. fournieri. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hormones in zygotic and microspore embryos of Brassica napus

A number of studies have used microspore-derived embryos (MDEs) as amodel for examining a range of processes, including hormonal regulation ofembryo development. We examined the hormonal physiology of MDEs with theprimaryobjective of testing the validity of using the MDE system as a model forhormonally-regulated development in zygotic embryos, through late stages. To dothis we identified and quantified endogenous levels of abscisic acid (ABA),indole-3-acetic acid (IAA) and a number of gibberellins (GAs), includingGA₁₉, GA₂₀, GA₁ and GA₈ in bothMDEsand zygotic embryos. The presence of GA₁₉, together with itsC₁₉ metabolites indicates that the early-13 hydroxylation pathway isoperative in both embryo systems. Gibberellins A₄ and GA₉were also identified, thereby confirming the presence of the earlynon-hydroxylation pathway in B. napus MDEs and zygoticembryos. In general, the pattern of change of hormone (ABA, IAA, GA₁and GA₂₀) content per embryo through embryogenesis was similar forMDEs and zygotic embryos. Indole-3-acetic acid and GA₁ increased toamaximum at day 30 after culture (DAC) before decreasing. Abscisicacid levels increased to a maximum at day 35, and declined in zygoticembryos but not in MDEs. GA₂₀ increased to the final harvest atmaturity, or day 40. The absolute content (g/embryo) of each hormone, howeverwas appreciably lower (5- to 15- fold) in the MDEs. This was not the result ofdilution into surrounding medium for ABA or IAA; GA₁, however, didaccumulate in the medium. Although there were absolute quantitative differencesin the levels of IAA and ABA found in the two embryo systems, the similaritiesin the pattern of hormone changes suggests that the MDE system can serve as auseful model for examining the physiological roles of hormones duringembryogenesis.     

Interactive effects of α-NAA and UV-B radiation on the endogenous hormone contents and growth of Trichosanthes kirilowii Maxim seedlings

The impact of UV-B radiation on endogenous hormones in plants has recently drawn attention from researchers. The mechanism for reduced stem elongation by UV-B might be due to changes in the phytohormone levels, especially IAA, which plays a role in stem elongation. In this study, effects of UV-B radiation on Trichosanthes kirilowii Maxim (T. kirilowii) seedlings in greenhouse-grown plants were investigated. The results indicated that: (1) In comparison to controls, exposure to 0.029 Jm^?2 s^?1. UV-B radiation led to accumulation of endogenous abscisic acid (ABA) and zeatinriboside (ZR) in the plant contents, and decreased contents of endogenous indole-3-acetic acid (IAA) and gibberellic acid (GA_1/3). Exposure to UV-B radiation reduced the height and leaf area of plants. As a result, total biomass (plant dry weight) was lower. (2) In comparison to controls, addition of 2 mg l^?1 α-naphthaleneacetic acid (α-NAA) slightly increased the contents of IAA, GA_1/3 and ZR, and decreased the content of ABA in leaves. This addition of α-NAA significantly increased plant height and leaf area, but only slightly increased total biomass. (3) Addition of α-NAA to UV-B-exposed plants: increased the content of endogenous IAA, GA_1/3 and ZR; decreased accumulation of endogenous ABA; and increased plant height and leaf area in comparison to plants that only were exposed to UV-B. Moreover, total biomass increased slightly. This suggests that addition of α-NAA may compensate to a certain extent for the lack of IAA resulting from UV-B radiation; it also increases the content of GA_1/3 and ZR, decreases the accumulation of ABA, and promotes the growth of plants.     

中国沙棘克隆生长对灌水强度的响应规律及其激素调控机制

关于中国沙棘克隆生长调节研究目前局限于外在机制,旨在探讨其克隆生长对灌水强度的响应规律及其激素调控的内在机制。结果表明:随着灌水强度的增大,分株生长、克隆繁殖、克隆扩散能力先升后降,IAA(吲哚-3-乙酸)、ZR(玉米素核苷)、GA_3(赤霉酸)含量及其与ABA(脱落酸)的比值先升后降而ABA含量先降后升。同时,分株生长、克隆繁殖、克隆扩散能力与IAA、ZR、GA_3含量以及IAA/ABA、ZR/ABA、GA_3/ABA呈极显著或显著正相关,与ABA含量呈极显著负相关。灌水强度过小或过大,IAA、ZR、GA_3含量以及IAA/ABA、ZR/ABA、GA_3/ABA低而ABA含量高,克隆生长潜力受到抑制,种群以分株小、数量少(分布稀疏)、扩散(水平根延伸和分枝)能力弱为特征,克隆生长格局倾向于"游击型"、种群早衰概率高;灌水强度适宜,IAA、ZR、GA_3含量以及IAA/ABA、ZR/ABA、GA_3/ABA高而ABA含量低,克隆生长潜力得以充分发挥,种群以分株大、数量多(分布密集)、扩散能力强为特征,克隆生长格局倾向于"聚集型"、种群稳定性高。随着灌水强度过小-适宜-过大的连续变化,中国沙棘通过改变激素状况调控克隆生长,从而形成与灌水强度相适应的克隆生长格局连续体"游击型-聚集型-游击型",种群稳定性呈"低-高-低"的连续变化过程。由此可见:灌水强度诱导内源激素发生改变,激素特征调控克隆生长格局,克隆生长格局决定种群稳定性。     

菰黑粉菌的分离鉴定及其发酵液中植物激素的检测

在显微镜下观察孢子形态,并观察单菌落的形态和菌株的微观形态,PCR扩增其ITS-5.8S rDNA序列,测序并在NCBI中进行同源比较,确定其种属。液体培养该菌株,通过高效液相色谱法检测发酵液中植物激素。结果表明：用菌落形态与孢子形态鉴定和分子生物学鉴定的方法,对茭白中分离的一个菌株鉴定为菰黑粉菌,且在其发酵液中检测到植物激素IAA、ABA和GA3,其中IAA含量为0.1306mg/L,ABA含量为0.01367mg/L。     

Hormonal changes associated with fruit set and development in mandarins differing in their parthenocarpic ability

Satsuma [Citrus unshiu (Mak) Marc.] and Clementine [Citrus reticulata (Hort.) Ex. Tanaka, cv. Oroval] are two related species of seedless mandarins which differ in their tendency to set parthenocarpic fruits. Satsuma fruits naturally set parthenocarpically whereas Clementine mandarins show very low ability to set fruit in the absence of cross-pollination. The endogenous levels of gibberellins (GAs) and free and conjugated indole-acetic acid (IAA) and abscisic acid (ABA) throughout early stages of fruit development were investigated in seedless cultivars of both species. Analyses performed by full-scan combined gas chromatography-mass spectrometry (GC-MS) of extracts from ovaries at anthesis demonstrated the presence of GA₁₉, GA₂₀, GA₂₉, GA₁, GA₈, GA₃ and iso-GA₃ in Satsuma mandarin, whereas only GA₂₉, GA₃ and trace levels of GA₈ were detected in Clementine. At this developmental stage GA-like substances, as estimated by bioassay, reached their highest levels in Satsuma, while Clementine mandarins contained relatively lower levels. In both species the highest levels of free IAA were found at petal-fall stage at which time free ABA levels also peaked. Developing fruits of Clementine had higher amounts of both free IAA and ABA. In Satsuma, levels of conjugated IAA remained low throughout reproductive development whereas in Clementine they increased as the free form declined. In contrast, conjugated ABA was at low levels in Clementine but reached higher concentrations in Satsuma. These results suggest that in these mandarins the potential for setting parthenocarpic fruits is mainly influenced by the hormonal status of the fruit during the later stages of cell division and early stages of cell enlargement. Thus, the condition of low ability to set parthenocarpic fruits appears to be associated with lower levels of active GAs, lower capability to catabolize ABA to conjugated ABA and higher ability to conjugate IAA during this period.     

Interrelationship between ethylene and growth regulators in the senescence of lettuce leaf discs

The interrelationship between ethylene and growth regulators in the senescence of romaine lettuce (Lactuca sativa L.) leaves was studied. Gibberellic acid (GA₃), kinetin, and 3-indoleacetic acid (IAA) retarded chlorophyll loss from leaf discs which were floated on hormone solutions. Abscisic acid (ABA) and ethephon enhanced chlorophyll loss and antagonized the senescence-retarding effect of GA₃ and kinetin. A high concentration of IAA (10^–4 M) caused accelerated chlorophyll loss, whereas a similar concentration of kinetin neither retarded nor promoted chlorophyll loss. The ineffectiveness of IAA and kinetin at their supraoptimal concentrations in retarding leaf senescence was related to increased production of ethylene induced in the treated leaf discs. GA₃ was the most effective in retarding chlorophyll loss and did not stimulate ethylene production at all. The senescence-enhancing effect of ABA was not mediated by ethylene. However, the moderately increased production of ethylene, induced by relatively high concentrations of ABA, could act synergistically with the latter to accelerate chlorophyll loss. It is proposed that the effectiveness of exogenously applied hormones, both in enhancing and retarding senescence, is greatly affected by the endogenous ethylene concentration of the treated plant tissue.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 2571-E, 1988 series.     

Effects of elevated carbon dioxide and/or ozone on endogenous plant hormones in the leaves of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Four-year-old Gingko (Ginkgo biloba L.) trees were exposed to ambient and elevated concentrations of CO₂ and O₃, and their combination for 1 year, using open-top chambers in Shenyang, China in 2006. Growth parameters and endogenous plant hormones were measured simultaneously over the experiment period. Elevated CO₂ increased leaf area and leaf dry weight but had no effect on shoot length, increased indole-3-acetic acid (IAA), gibberellins A₃ (GA₃), zeatin riboside (ZR), dihydrozeatin (DHZR) and isopentenyl-adenosine (iPA) content but decreased abscisic acid (ABA) content. Elevated O₃ significantly decreased leaf area, leaf dry weight, shoot length, and decreased IAA, GA₃, ZR content but increased ABA content and had a little effect on iPA, DHZR content. Elevated CO₂ + O₃ decreased IAA, iPA and DHZR content while increased ABA and GA₃ content in the early stage of the exposure and then decreased in the late stage. The evidence from this study indicates that elevated CO₂ ameliorated the effects of elevated ozone on tree growth, and elevated CO₂ may have a largely positive impact on forest tree growth while elevated O₃ will likely have a negative impact.