Isolation of Indole-3-Acetyl Amino Acids using Polyvinylpolypyrrolidone Chromatography

Amino acid conjugates of IAA can be chromatographed on polyvinylpolypyrrolidone (PVPP) using either acidic methanol or aqueous buffers as eluents. In the aqueous system, elution of the compounds is pH-dependent, and the pattern obtained suggests that hydrophobic interactions contribute substantially to the chromatographic behavior of IAA peptides on PVPP. Purification of soybean seed extracts by PVPP chromatography produced fractions containing indole-3-acetylaspartate and indole-3-acetylglutamate, based on chromatographic and mass spectral analysis, as well as three other indolic compounds, tentatively identified as N-acyl tryptophan derivatives. PVPP chromatography provides an effective preliminary purification of IAA peptides from plant extracts prior to their separation by other techniques such as high performance liquid chromatography.     

利用高效液相色谱法分离和测定棉花组织培养过程中4种内源激素

目的：优化利用高效液相色谱法测定棉花组织培养过程中吲哚-3-乙酸（IAA）、玉米素（ZT）、赤霉素（GA3）和脱落酸（ABA）等4种植物内源激素的条件,了解棉花胚性愈伤组织发生过程中4种内源激素及激素含量比例的规律性变化,以及添加不同外源激素对内源激素和愈伤组织的影响,为棉花组织培养由经验型变为理论型提供基础。方法：采用高效液相色谱法。结果：棉花胚性愈伤组织发生过程中4种内源激素及激素含量比例呈规律性变化：ZT、ABA、ABA/GA3、ABA/IAA呈现先上升后下降的趋势,GA3呈现先上升后下降再上升的趋势,ZT/IAA呈现明显的上升-下降-上升-下降的趋势;不同激素组合诱导下愈伤组织中内源激素含量及愈伤组织状态也有较大差别。结论：研究结果对指导组织培养过程中激素的调整和配比、制定适宜的培养计划有一定的指导意义;4种不同的内源激素都是愈伤组织生长的重要因子,各自适宜的浓度和恰当的比例调节着外植体的脱分化和再分化,各种激素协同作用促进细胞的生长和分化。     

Capsaicinoid Formation in the Protoplast from the Placenta of Capsicum Fruits

Sixty-two phenylacetic acid derivatives having phenylpropane skeleton were synthesized. Their physiological effects on the growth of lettuce seedlings were distinguished from those of plant hormones and an antiauxin. The derivatives retarded the growth in a similar manner to the auxin transport inhibitors, NPA and CPD. A few derivatives including 2-[3′-(m-chlorophenyl)isoxazol-5′-yl]phenylacetic acid were up to 10 times as potent as CPD in inhibition of the hypocotyl growth and IAA transport. The phenylacetic acids did not cause, even at high concentrations, any serious damage such as a herbicidal would. Their remarkable growth-retarding effects may be due, at least in part, to their ability to prevent the polar movement of IAA within tissues.     

Free radical–scavenging activity and DNA damaging potential of auxins IAA and 2‐methyl‐IAA evaluated in human neutrophils by the alkaline comet assay

Auxins, of which indole‐3‐acetic acid (IAA) is the most widespread representative, are plant hormones. In addition to plants, IAA also naturally occurs in humans in micromolar concentrations. In the presence of peroxidase, indolic auxins are converted to cytotoxic oxidation products and have thus been proposed for use in gene‐directed enzyme/prodrug tumor therapy. Since data on the genotoxicity of IAA and its derivatives are not consistent, here we investigate the early DNA damaging effects (2‐h treatment) of the auxins, IAA, and 2‐methyl‐indole‐3‐acetic acid (2‐Me‐IAA) by the alkaline comet assay and compare them with their free radical–scavenging activity measured by the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assay. Human neutrophils are chosen as the test system since they possess inherent peroxidase activity. The results of the comet assay indicate an increase in DNA damage in a dose‐dependent manner up to 1.00 mM of both auxins. Generally, IAA applied in the same concentration had greater potential to damage DNA in human neutrophils than did 2‐Me‐IAA. The genotoxicities of the two examined auxins are negatively correlated with their antioxidant activities, as measured by the DPPH assay; 2‐Me‐IAA showed a higher antioxidant capacity than did IAA. We assume that differences in the molecular structure of the tested auxins contributed to differences in their metabolism, in particular, with respect to interactions with peroxidases and other oxidative enzymes in neutrophils. However, the exact mechanisms have to be elucidated in future studies. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:165–173, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20323     

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。     

三种外源植物激素对黄花风铃木幼苗生物量、叶绿素荧光参数及光合特性的影响

以黄花风铃木(Tabebuia chrysantha (Jacq.) Nichols)幼苗为材料,采用L16(45)正交实验方法,测定生根粉(GGR)、3-吲哚乙酸(IAA)、多效唑(PP333) 3种外源植物激素不同组合处理下黄花风铃木幼苗的生物量、叶绿素含量、叶绿素荧光参数以及光合特性,研究3种外源植物激素不同组合对黄花风铃木幼苗生长和光合作用的影响,筛选出适宜黄花风铃木幼苗生长的最佳浓度组合。结果显示:11号处理(GGR 400 mg/L、IAA 400 mg/L、PP333400 mg/L)幼苗鲜重、干重均大于其他组合处理。3种外源植物激素中,GGR对幼苗鲜重、幼苗干重增长起主导作用,IAA和PP333的作用不明显。11号处理下的幼苗净光合速率、气孔导度、蒸腾速率、叶绿素含量、叶绿素荧光参数均大于其他处理,胞间二氧化碳浓度值最低。分析得出IAA对黄花风铃木幼苗的净光合速率、气孔导度、蒸腾速率、光合系统潜在活性、最大光能转换效率和叶绿素含量起主导作用,GGR和PP333的作用不明显。综合分析各指标得出,适宜黄花风铃木幼苗生长的最佳外源激素浓度组合为11号处理,此浓度下黄花风铃木幼苗综合质量最佳。     

Comparison of effects of ABA and IAA on phospholipid bilayers

The plant hormones indole-3-acetic acid (IAA) and abscisic acid (ABA) affect the properties of phospholipid bilayers differently. IAA enhances permeability of bilayers composed of phosphatidylcholine to the non-electrolyte erythritol while ABA requires an additional phospholipid in the membrane to produce substantial enhancement. Similar conclusions are obtained by measuring hormone-induced permeability to chloride ions; IAA is effective with single component phosphatidylcholine membranes while ABA requires a second phospholipid. Erythritol permeability is shown to be pH dependent for both hormones. Although IAA is more effective at increasing erythritol permeability at pH 4 than at pH 7, both dissociated and undissociated IAA affect the process. In comparison ABA is almost totally ineffective in the dissociated form (at pH 7). Spin label electron spin resonance measurements demonstrated that neither hormone substantially disrupts acyl chain mobility within the membrane, indicating that the mechanism of permeability enhancement is not a general non-specific pertubation of membrane ordering and fluidity. Both hormones can also effect the stability of small unilamellar (sonicated) vesicles. Phosphatidylcholine vesicles are relatively stable and do not rapidly aggregate with either ABA or IAA. However, when phosphatidylethanolamine is incorporated as a minor component (10 mol%) into phosphatidylcholine vesicles ABA causes rapid aggregation while IAA has no effect. These experiments indicate that the two hormones may exhibit completely different behaviour on membranes without the requirement for specific proteinaceous receptors.     

Dynamic Changes of Endogenous Phytohormones During the Development of Embryos Derived from Wheat Crossed with Zea mays and Tripsacum dactyloides

The embryos derived from intergeneric crosses between Triticum aestivum L. (2n = 6x= 42) and Zea mays L. (2n= 20), Tripsacum dactyloides L. (2n= 4x= 72), as results of the elimination of paternal chromosome and having no endosperm as normal, are difficult to develop completely in vivo. Hormonal analyses in such intergeneric hybridized ovaries have been carried out including indole-3-acetic acid (IAA), zeatin (Z) and zeatin riboside (ZR), and abscisic acid (ABA) during different developmental stages of embryo. The results indicated that IAA level in hybridized ovaries was much lower than that in self-pollinated ovaries. Moreover, the active changes of Z+ZR and ABA contents were also different between the above two types of ovary. Nevertheless, the ratio changes of IAA/(Z+ZR) and IAA/ABA were similar between the two types of ovary, the same was true in the ratio changs of IAA, Z+ZR, and ABA to the sum of these three types of endogenous hormones. But the three types of ratios in hybridized ovaries were varied far more greater than those in self-pollinated ones. It suggested the importance to maintain dynamic equilibrium of multiple hormones and sequential regulation during the embryo development. That the distantly hybridized embryos failed to mature successfully was probably associated with a deviation from the normal changes of endogenous hormones levels and balance system of multiple hormones in addition to the lack of endosperm nourishment.     

Analysis of 3-indolylacetic acid and abscisic acid by high-performance liquid chromatography and gas-liquid chromatography

A method of analysis of 3-indolylacetic acid (IAA) and abscisic acid (ABA), allowing the simultaneous extraction of both regulators from plant material, has been developed. The method involves extraction with methanol, isolation of the acid fraction, diazomethane methylation, separation of the hormones through reverse-phase preparative high-performance liquid chromatography, and quantification of both compounds by gas-liquid chromatography. The recovery percentage at each step was monitored with radioactive compounds added at the beginning of the process. The final recovery was 70% for IAA and 96% for ABA. The method was applied to the analysis of the IAA and ABA content of stems of hazel (Corylus avellana L.).     

金塔柏扦插不定根形成与内源激素的调控研究

金塔柏（Platycladus orientalis ‘Beverleyensis’）是重要的观赏树种。生长素（IAA）、玉米素（ZT）、脱落酸（ABA）和茉莉酸（JA）在金塔柏扦插不定根再生过程中起着重要的调控作用,但不同发育阶段内源激素的动态变化及其对不定根发生的影响仍不清楚。以金塔柏半木质化枝条为材料,采用连续组织切片技术观察了不定根发生过程,利用高效液相色谱串联质谱法检测了4种内源激素含量的动态变化。结果表明,金塔柏不定根原基起源于愈伤组织、髓射线、木质部、维管形成层、次生韧皮部、皮层、髓射线与形成层交界处等部位,属于多位点发生模式和多类型生根方式。在不定根形成过程中,随着愈伤组织的形成,IAA和ZT含量下降,ABA和JA含量升高;随着根原基的分化,IAA和ZT含量缓慢升高,ABA和JA含量下降;随着不定根形成与伸长,IAA、ZT、JA逐渐升高,ABA维持在低水平。激素平衡分析发现,IAA/ABA比值和IAA/JA比值下降、IAA/ZT比值上升利于愈伤组织的形成,反之利于根原基的诱导分化,而IAA/ABA比值升高,IAA/ZT和IAA/JA维持在较低水平利于不定根形成与伸长。研究结果为揭示不同内源激素对金塔柏扦插不定根再生的调节作用提供了依据。     

Concentration of Indole-3-acetic Acid and Its Derivatives in Plants

Seeds of oat, coconut, soybean, sunflower, rice, millet, kidney bean, buckwheat, wheat, and corn and vegetative tissue of oat, pea, and corn were assayed for free indole-3-acetic acid (IAA), esterified IAA, and peptidyl IAA. Three conclusions were drawn: (a) all plant tissues examined contained most of their IAA as derivatives, either esterified or as a peptide; (b) the cereal grains examined contained mainly ester IAA; (c) the legume seeds examined contained mainly peptidyl IAA. Errors in analysis of free and bound IAA are discussed.     

The Fate of Exogenously Applied Indoleacetic Acid in Light Grown Stems

Elongating segments from light grown pea (Pisum sativum L. cv. Alaska) and bean (Pbaseolus vulgaris L. cv. Red kidney) stems were incubated in 10^-5M indoleacetic acid (IAA)-1-¹⁴C,and -5-³H in the light. Radoactive derivatives, extracted in water, ethanol or ether, and 1 N sodium hydroxide at three different times were chromatographed in three separate systems and the different metabolites identified by their labeling and chromatographic characteristics. Major metabolites included indoleacetyl aspartate, possibly indoleacetyl glucoside, hydroxymethyloxindole, and in bean a further major unidentified compund. Other compounds occurred in lesser amounts. Indole aldehyde was present only in very small quantities. IAA breakdown commenced between 1 and 6 h of incubation, following which IAA decreased and most metabolites increased, though IAA was still present after 24 h. Alkaline hydrolysates contained mainly IAA at a level which changed little between 6 and 24 h.     

棉花胚珠和叶片激素含量与纤维性状的相关分析

对具有同一遗传背景的 35份变异材料 ,在特定生育时期 ,进行了叶片和胚珠内源激素含量与其纤维性状的典型相关和主成分分析 .结果表明 ,棉花激素与纤维性状间存在一定的相关关系 ,胚珠和叶片激素含量与纤维性状的相关关系存在较大差异 .在胚珠中 ,IAA、ABA、GAs、ZRs 4种激素与纤维性状的相关关系中 ,ABA与衣分的显著正相关最重要 ,而在叶片中 ,GAs与纤维麦克隆值的极显著正相关则起着决定性的作用 .激素和纤维性状的主成分分析结果表明 ,第一主成分与激素有关 ,胚珠中的主要因子是 IAA和 ABA,叶片中则是 ABA、ZRs、GAs,第二主成分中最重要的因子为纤维强度和长度     

牡丹冬季室内催花过程中内源激素含量的变化

牡丹品种朱砂垒(PaeoniasuffruticosaAndr.cv.Zhushalei)在冬季室内催花过程中7种内源激素含量变化不同。玉米素核苷(Z ZR)、生长素(IAA)和赤霉素(GA3)的含量在花生长发育过程中处于较高水平;而脱落酸(ABA)、异戊烯基腺苷(IP IPA)、二氢玉米素核苷(DHZ DHZR)、赤霉素(GA4)的含量低于上述3种内源激素。激素平衡方面,GAs/ABA、CTKs/ABA、IAA/ABA处于较高水平,变化幅度较大。在催花过程中,内源激素以及其平衡影响牡丹花的生长发育。本研究结果对牡丹花期调控提供理论依据。     

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.     

A Quantitative Estimation of Alkali-labile Indole-3-Acetic Acid Compounds in Dormant and Germinating Maize Kernels

An estimate has been made of the quantities of alkali-labile esters of indoleacetic acid (IAA) in kernels of sweet corn (Zea mays). The amount is between 70 to 90 mg of IAA per kilogram of dry kernels. About one-half of the IAA is present as high molecular weight esters and the remaining one-half as esters of myo-inositol. Free IAA, which may have existed in the kernels, or may have resulted from ester hydrolysis during isolation or storage, amounts to between 1 to 10% of the esterified IAA. Five newly observed low molecular weight indoleacetyl compounds are described and their chromatographic behavior reported. The total IAA content of corn kernels and intact seedlings decreases during germination, declining to about 10% of the original content during 96 hr of germination. Difficulties in obtaining quantitative results and the possible physiological significance of these results is discussed.     

Electric and structural studies of hormone interaction with chloroplast envelope membranes isolated from vegetative and generative rape

The electric and structural properties of envelope membranes of chloroplasts obtained from vegetative and generative plants of rape and the effect of hormone (IAA, GA(3) and zearalenone) treatment were determined by zeta potential and small-angle X-ray scattering (SAXS) methods. Chloroplasts were isolated from leaves cut off from the vegetative (before cooling) and generative apical parts of plants. The lipid composition of chloroplast envelope membranes were analyzed by chromatographic techniques. Envelopes from generative plants contained higher levels of digalactosyldiacylglycerol (DGDG) and smaller amounts of phospholipids (PLs) in comparison to those obtained from vegetative ones. Moreover, envelopes of generative plants were characterized by higher fractions of unsaturated fatty acids. The zeta potential changes caused by hormone treatment were higher for chloroplasts isolated from vegetative plants in comparison to chloroplasts isolated from generative ones. An especially strong effect was observed for chloroplasts treated with IAA. The thickness of bilayers of untreated chloroplasts from vegetative plants were larger by 0.4 nm when comparing to the thickness of layers obtained from generative ones. The effect of hormones (GA(3) and zearalenone) was detected only for vegetative chloroplasts. Both applied methods indicated differences in the properties of untreated and hormone-treated chloroplasts obtained from vegetative and generative plants.     

The Effect of Applied Growth Hormones on Cambial Division and the Differentiation of the Cambial Derivatives

When indole-3-acetic acid (IAA) is applied to woody shoots cambialdivision is stimulated and the cambial derivatives differentiateto produce xylem tissue. When gibberellic acid (GA) is applied,cambial division occurs but the resultant derivatives on thexylem side of the cambium remain undifferentiated. The relativelevels of applied IAA and GA are important in determining whethermainly xylem or phloem tissue is produced. High IAA/low GA concentrationsfavour xylem formation, whereas low IAA/high GA concentrationsfavour phloem production. The new phloem tissue produced asa result of hormone treatment is fully differentiated, containingsieve elements and sieve plates. IAA is important in promotingthe elongation of the cambial derivatives to produce xylem vesseland fibre elements, though in the case of xylem fibres appliedGA causes further elongation. IAA is an important factor indetermining vessel diameter in the ring-porous species Robiniapseudacacia, high levels of applied IAA giving wide springwood-typevessels and low levels giving narrow ‘summerwood’vessels.